Compounds and methods for delivery of prostacyclin analogs

ABSTRACT

This invention pertains generally to prostacyclin analogs and methods for their use in promoting vasodilation, inhibiting platelet aggregation and thrombus formation, stimulating thrombolysis, inhibiting cell proliferation (including vascular remodeling), providing cytoprotection, preventing atherogenesis and inducing angiogenesis. Generally, the compounds and methods of the present invention increase the oral bioavailability and circulating concentrations of treprostinil when administered orally. Compounds of the present invention have the following formula:

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.11/189,072, filed Jul. 26, 2005, which is a continuation of U.S.application Ser. No. 10/851,481, filed May 24, 2004, now U.S. Pat. No.7,417,070, which claims benefit of U.S. Provisional Application Ser. No.60/472,407, filed on May 22, 2003, the entire contents of whichapplications are incorporated by reference herein.

FIELD OF THE INVENTION

This invention pertains generally to prostacyclin analogs and methodsfor their use in promoting vasodilation, inhibiting platelet aggregationand thrombus formation, stimulating thrombolysis, inhibiting cellproliferation (including vascular remodeling), providing cytoprotection,preventing atherogenesis and inducing angiogenesis. Through theseprostacyclin-mimetic mechanisms, the compounds of the present inventionmay be used in the treatment of/for: pulmonary hypertension, ischemicdiseases (e.g., peripheral vascular disease, Raynaud's phenomenon,Scleroderma, myocardial ischemia, ischemic stroke, renal insufficiency),heart failure (including congestive heart failure), conditions requiringanticoagulation (e.g., post MI, post cardiac surgery), thromboticmicroangiopathy, extracorporeal circulation, central retinal veinocclusion, atherosclerosis, inflammatory diseases (e.g., COPD,psoriasis), hypertension (e.g., preeclampsia), reproduction andparturition, cancer or other conditions of unregulated cell growth,cell/tissue preservation and other emerging therapeutic areas whereprostacyclin treatment appears to have a beneficial role. Thesecompounds may also demonstrate additive or synergistic benefit incombination with other cardiovascular agents (e.g., calcium channelblockers, phosphodiesterase inhibitors, endothelial antagonists,antiplatelet agents).

BACKGROUND OF THE INVENTION

Many valuable pharmacologically active compounds cannot be effectivelyadministered orally for various reasons and are generally administeredvia intravenous or intramuscular routes. These routes of administrationgenerally require intervention by a physician or other health careprofessional, and can entail considerable discomfort as well aspotential local trauma to the patient.

One example of such a compound is treprostinil, a chemically stableanalog of prostacyclin. Although treprostinil sodium (Remodulin®) isapproved by the Food and Drug Administration (FDA) for subcutaneousadministration, treprostinil as the free acid has an absolute oralbioavailability of less than 10%. Accordingly, there is clinicalinterest in providing treprostinil orally.

Thus, there is a need for a safe and effective method for increasing thesystemic availability of treprostinil via administration of treprostinilor treprostinil analogs.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compound havingstructure I:

wherein,

R¹ is independently selected from the group consisting of H, substitutedand unsubstituted benzyl groups, and groups wherein OR¹ are substitutedor unsubstituted glycolamide esters;

R² and R³ may be the same or different and are independently selectedfrom the group consisting of H, phosphate and groups wherein OR² and OR³form esters of amino acids or proteins, with the proviso that all of R¹,R² and R³ are not H;

an enantiomer of the compound;

and pharmaceutically acceptable salts of the compound and polymorphs.

In some of these embodiments, R¹ is a substituted or unsubstitutedbenzyl group, such as CH₂C₆H₅. In other embodiments, OR¹ is asubstituted or unsubstituted glycolamide ester, R¹ is —CH₂CONR⁴R⁵, R⁴and R⁵ may be the same or different and are independently selected fromthe group consisting of H, OH, substituted and unsubstituted alkylgroups, —(CH₂)_(m)CH₃, —CH₂OH, and —CH₂(CH₂)_(n)H, with the proviso thatm is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3 or 4. In certain of theseembodiments one or both of R⁴ and R⁵ are independently selected from thegroup consisting of H, —OH, —CH₃, or —CH₂CH₂OH. In any of the previouslydiscussed embodiments, one or both of R² and R³ can be H. In someenantiomers of the compound R¹═R²═R³═H, or R²═R³═H and R¹=valinyl amide.

In still further embodiments of the present compounds R² and R³ areindependently selected from phosphate and groups wherein OR² and OR³ areesters of amino acids, dipeptides, esters of tripeptides and esters oftetrapeptides. In some compounds only one of R² or R³ is a phosphategroup. In other compounds R² and R³ are independently selected fromgroups wherein OR² and OR³ are esters of amino acids, such as esters ofglycine or alanine. In any of the above embodiments, one of R² and R³are H. In certain of the present compounds, the oral bioavailability ofthe compound is greater than the oral bioavailability of treprostinil,such as at least 50% or 100% greater than the oral bioavailability oftreprostinil. The above compounds can further comprise an inhibitor ofp-glycoprotein transport. Any of these compounds can also furthercomprise a pharmaceutically acceptable excipient.

The present invention also provides a method of using the abovecompounds therapeutically of/for: pulmonary hypertension, ischemicdiseases, heart failure, conditions requiring anticoagulation,thrombotic microangiopathy, extracorporeal circulation, central retinalvein occlusion, atherosclerosis, inflammatory diseases, hypertension,reproduction and parturition, cancer or other conditions of unregulatedcell growth, cell/tissue preservation and other emerging therapeuticareas where prostacyclin treatment appears to have a beneficial role. Apreferred embodiment is a method of treating pulmonary hypertensionand/or peripheral vascular disease in a subject comprising orallyadministering a pharmaceutically effective amount of a compound ofstructure II:

wherein,

R¹ is independently selected from the group consisting of H, substitutedand unsubstituted alkyl groups, arylalkyl groups and groups wherein OR¹form a substituted or unsubstituted glycolamide ester;

R² and R³ may be the same or different and are independently selectedfrom the group consisting of H, phosphate and groups wherein OR² and OR³form esters of amino acids or proteins, with the proviso that all of R¹,R² and R³ are not H;

an enantiomer of the compound; and

a pharmaceutically acceptable salt or polymorph of the compound.

In some of these methods, when OR¹ forms a substituted or unsubstitutedglycolamide ester, R¹ is —CH₂CONR⁴R⁵, wherein R⁴ and R⁵ may be the sameor different and are independently selected from the group consisting ofH, OH, substituted and unsubstituted alkyl groups, —(CH₂)_(m)CH₃,—CH₂OH, and —CH₂(CH₂)_(n)H, with the proviso that m is 0, 1, 2, 3 or 4,and n is 0, 1, 2, 3 or 4. In other methods R¹ is a C₁-C₄ alkyl group,such as methyl, ethyl, propyl or butyl. In the disclosed methods, R¹ canalso be a substituted or unsubstituted benzyl group. In other methods,R¹ can be —CH₃ or —CH₂C₆H₅. In still other methods R⁴ and R⁵ are thesame or different and are independently selected from the groupconsisting of H, OH, —CH₃, and —CH₂CH₂OH. In yet other methods, one orboth of R² and R³ are H. Alternatively, one or both of R² and R³ are notH and R² and R³ are independently selected from phosphate and groupswherein OR² and OR³ are esters of amino acids, dipeptides, esters oftripeptides and esters of tetrapeptides. In some methods, only one of R²or R³ is a phosphate group. In additional methods, R² and R³ areindependently selected from groups wherein OR² and OR³ are esters ofamino acids, such as esters of glycine or alanine. In further methodsone of R¹ and R² is H. In some methods, enantiomers of the compoundwhere R¹═R²═R³═H, or R²═R³═H and R¹=valinyl amide are used.

In various methods the oral bioavailability of the compound is greaterthan the oral bioavailability of treprostinil, such as at least 50% or100% greater than the oral bioavailability of treprostinil. The presentmethods can also comprise administering pharmaceutically effectiveamount of a p-glycoprotein inhibitor, simultaneously, sequentially, orprior to administration of the compound of structure II. In someembodiments the p-glycoprotein inhibitor is administered orally orintravenously. The disclosed methods can be used to treat pulmonaryhypertension.

The present invention also provides a method of increasing the oralbioavailability of treprostinil or pharmaceutically acceptable saltthereof, comprising administering a pharmaceutically effective amount ofa p-glycoprotein inhibitor and orally administering a pharmaceuticallyeffective amount of treprostinil to a subject. In certain of theseembodiments the p-glycoprotein inhibitor is administered prior to orsimultaneously with the treprostinil. The route of the p-glycoproteininhibitor administration can vary, such as orally or intravenously. Thepresent invention also provides a composition comprising treprostinil ora pharmaceutically acceptable salt thereof and a p-glycoproteininhibitor.

The present compound can also be administered topically ortransdermally.

Pharmaceutical formulations according to the present invention areprovided which include any of the compounds described above incombination with a pharmaceutically acceptable carrier.

The compounds described above can also be used to treat cancer.

Further objects, features and advantages of the invention will beapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B respectively show plasma concentration versus timecurves for intravenous and intraportal dosing of treprostinildiethanolamine salt in rats as described in Example 1;

FIGS. 2A, 2B and 2C respectively show plasma concentration versus timecurves for intraduodenal, intracolonic and oral dosing of treprostinildiethanol amine salt in rats as described in Example 1;

FIG. 3 shows on a logarithmic scale the average plasma concentrationversus time curves for the routes of administration described in Example1;

FIG. 4 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following oral administration in ratsof treprostinil methyl ester as described in Example 2;

FIG. 5 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following oral administration in ratsof treprostinil benzyl ester as described in Example 2;

FIG. 6 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following oral administration in ratsof treprostinil diglycine as described in Example 2;

FIG. 7 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following oral administration inrates of treprostinil benzyl ester (0.5 mg/kg) and treprostinildiglycine (0.5 mg/kg) as described in Example 2 compared to treprostinil(1 mg/per kg).

FIG. 8 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following intraduodenaladministration of treprostinil monophosphate (ring) as described inExample 3;

FIG. 9 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following intraduodenaladministration of treprostinil monovaline (ring) as described in Example3;

FIG. 10 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following intraduodenaladministration of treprostinil monoalanine (ring) as described inExample 3;

FIG. 11 is a graphical representation of the plasma concentration versustime curve for treprostinil in rat following intraduodenaladministration of treprostinil monoalanine (chain) as described inExample 3; and

FIG. 12 is a graphical representation of the average plasmaconcentration versus time curve for each prodrug compared totreprostinil alone from Example 1, as described in Example 3.Treprostinil was dosed at 1 mg/kg whereas the prodrugs were dosed at 0.5mg/kg.

FIGS. 13A-13D respectively show doses, administered every two hours forfour doses, for either 0.05 mg per dose (total=0.2 mg), 0.125 mg perdose (total=0.5 mg), 0.25 mg per dose (total=1.0 mg), or 0.5 mg per dose(total=2.0 mg).

FIG. 14 shows pharmacokinetic profiles of UT-15C sustained releasetablets and sustained release capsules, fasted and fed state.

FIG. 15 shows an X ray powder diffraction spectrum of the polymorph FormA.

FIG. 16 shows an IR spectrum of the polymorph Form A.

FIG. 17 shows a Raman spectrum of the polymorph Form A.

FIG. 18 shows thermal data of the polymorph Form A.

FIG. 19 shows moisture sorption data of the polymorph Form A.

FIG. 20 shows an X ray powder diffraction spectrum of the polymorph FormB.

FIG. 21 shows thermal data of the polymorph Form B.

FIG. 22 shows moisture sorption data of the polymorph Form B.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, “a” or “an” means “one or more.” The presentinvention provides compounds and methods for inducing prostacyclin-likeeffects in a subject or patient. The compounds provided herein can beformulated into pharmaceutical formulations and medicaments that areuseful in the methods of the invention. The invention also provides forthe use of the compounds in preparing medicaments and pharmaceuticalformulations and for use of the compounds in treating biologicalconditions related to insufficient prostacyclin activity as outlined inthe Field of Invention. The present invention also provides compoundsand methods for the treatment of cancer and cancer related disorders.

In some embodiments, the present compounds are chemical derivatives of(+)-treprostinil, which has the following structure:

Treprostinil is a chemically stable analog of prostacyclin, and as suchis a potent vasodilator and inhibitor of platelet aggregation. Thesodium salt of treprostinil,(1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-Hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]aceticacid monosodium salt, is sold as a solution for injection as Remodulin®which has been approved by the Food and Drug Administration (FDA) fortreatment of pulmonary hypertension. In some embodiments, the presentcompounds are derivatives of (−)-treprostinil, the enantiomer of(+)-treprostinil. A preferred embodiment of the present invention is thediethanolamine salt of treprostinil. The present invention furtherincludes polymorphs of the above compounds, with two forms, A and B,being described in the examples below. Of the two forms, B is preferred.A particularly preferred embodiment of the present invention is form Bof treprostinil diethanolamine.

In some embodiments, the present compounds are generally classified asprodrugs of treprostinil that convert to treprostinil afteradministration to a patient, such as through ingestion. In someembodiments, the prodrugs have little or no activity themselves and onlyshow activity after being converted to treprostinil. In someembodiments, the present compounds were produced by chemicallyderivatizing treprostinil to make stable esters, and in some instances,the compounds were derivatized from the hydroxyl groups. Compounds ofthe present invention can also be provided by modifying the compoundsfound in U.S. Pat. Nos. 4,306,075 and 5,153,222 in like manner.

In one embodiment, the present invention provides compounds of structureI:

wherein,

R¹ is independently selected from the group consisting of H, substitutedand unsubstituted benzyl groups and groups wherein OR¹ are substitutedor unsubstituted glycolamide esters;

R² and R³ may be the same or different and are independently selectedfrom the group consisting of H, phosphate and groups wherein OR² and OR³form esters of amino acids or proteins, with the proviso that all of R¹,R² and R³ are not H;

enantiomers of the compound; and

pharmaceutically acceptable salts of the compound.

In some embodiments wherein OR¹ are substituted or unsubstitutedglycolamide esters, R¹ is —CH₂CONR⁴R⁵ and R⁴ and R⁵ may be the same ordifferent and are independently selected from the group consisting of H,OH, substituted and unsubstituted alkyl groups, —(CH₂)_(m)CH₃, —CH₂OH,and —CH₂(CH₂)_(n)H, with the proviso that m is 0, 1, 2, 3 or 4, and n is0, 1, 2, 3 or 4.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group orthe groups described in the R of structures I and II above and below,the present invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention. For example, R¹ can specificallyexclude H, substituted and unsubstituted benzyl groups, or groupswherein OR¹ are substituted or unsubstituted glycolamide esters.

In some embodiments, R¹ is a substituted or unsubstituted benzyl groups,such as —CH₂C₆H₅, —CH₂C₆H₄NO₂, —CH₂C₆H₄OCH₃, —CH₂C₆H₄Cl, —CH₂C₆H₄(NO₂)₂,or —CH₂C₆H₄F. The benzyl group can be ortho, meta, para, ortho/parasubstituted and combinations thereof. Suitable substituents on thearomatic ring include halogens (fluorine, chlorine, bromine, iodine),—NO₂ groups, —OR¹⁶ groups wherein R¹⁶ is H or a C₁-C₄ alkyl group, andcombinations thereof.

Alternatively, when R¹ is —CH₂CONR⁴R⁵ then R⁴ and R⁵ may be the same ordifferent and are independently selected from the group consisting of H,OH, —CH₃, and —CH₂CH₂OH. In these compounds where R¹ is not H, generallyone or both of R² and R³ are H.

In some embodiment one or both of R² and R³ are H and R¹ is —CH₂CONR⁴R⁵,and one or both of R⁴ and R⁵ are H, —OH, —CH₃, —CH₂CH₂OH.

In compounds where one or both of R² and R³ are not H, R² and R³ can beindependently selected from phosphate and groups wherein OR² and OR³ areesters of amino acids, dipeptides, esters of tripeptides and esters oftetrapeptides. In some embodiments, only one of R² or R³ is a phosphategroup. In compounds where at least one of R² and R³ is not H, generallyR¹ is H. In additional embodiments, one of R² and R³ are H and thus thecompound of structure I is derivatized at only one of R² and R³. Inparticular compounds, R² is H and R³ is defined as above. In additionalembodiments, R¹ and R³ are H and R² is a group wherein OR² is an esterof an amino acid or a dipeptide. In further embodiments, R¹ and R² are Hand R³ is a group wherein OR³ is an ester of an amino acid or adipeptide.

When one or both of the OR² and OR³ groups form esters of amino acids orpeptides, i.e., dipeptides, tripeptides or tetrapeptides, these can bedepicted generically as —COCHR⁶NR⁷R⁸ wherein R⁶ is selected from thegroup consisting of amino acid side chains, R⁷ and R⁸ may be the same ordifferent and are independently selected from the group consisting of H,and —COCHR⁹NR¹⁰R¹¹. Generally, reference to amino acids or peptidesrefers to the naturally occurring, or L-isomer, of the amino acids orpeptides. However, the present compounds and methods are not limitedthereto and D-isomer amino acid residues can take the place of some orall of L-amino acids. In like manner, mixtures of D- and L-isomers canalso be used. In the embodiments wherein the amino acid is proline, R⁷together with R⁶ forms a pyrrolidine ring structure. R⁶ can be any ofthe naturally occurring amino acid side chains, for example —CH₃(alanine), —(CH₂)₃NHCNH₂NH (arginine), —CH₂CONH₂ (asparagine), —CH₂COOH(aspartic acid), —CH₂SH (cysteine), —(CH₂)₂CONH₂ (glutamine),—(CH₂)₂COOH (glutamic acid), —H (glycine), —CHCH₃CH₂CH₃ (isoleucine),—CH₂CH(CH₃)₂ (leucine), —(CH₂)₄NH₂ (lysine), —(CH₂)₂SCH₃ (methionine),—CH₂Ph (phenylalanine), —CH₂OH (serine), —CHOHCH₃ (threonine), —CH(CH₃)₂(valine),

—(CH₂)₃NHCONH₂ (citrulline) or —(CH₂)₃NH₂ (ornithine). Ph designates aphenyl group.

In the above compounds, R⁷ and R⁸ may be the same or different and areselected from the group consisting of H, and —COCHR⁹NR¹⁰R¹¹, wherein R⁹is a side chain of amino acid, R¹⁰ and R¹¹ may be the same or differentand are selected from the group consisting of H, and —COCHR¹²NR¹³R¹⁴,wherein R¹² is an amino acid side chain, R¹³ and R¹⁴ may be the same ordifferent and are independently selected from the group consisting of H,and —COCHR¹⁵NH₂. One skilled in the art will realize that the peptidechains can be extended on the following scheme to the desired length andinclude the desired amino acid residues.

In the embodiments where either or both of OR² and OR³ groups form anester of a peptide, such as dipeptide, tripeptide, tetrapeptide, etc.the peptides can be either homopeptides, i.e., repeats of the same aminoacid, such as arginyl-arginine, or heteropeptides, i.e., made up ofdifferent combinations of amino acids. Examples of heterodipeptidesinclude alanyl-glutamine, glycyl-glutamine, lysyl-arginine, etc.

As will be understood by the skilled artisan when only one R⁷ and R⁸includes a peptide bond to further amino acid, such as in the di, triand tetrapeptides, the resulting peptide chain will be linear. When bothR⁷ and R⁸ include a peptide bond, then the peptide can be branched.

In still other embodiments of the present compounds R¹ is H and one ofR² or R³ is a phosphate group or H while the other R² or R³ is a groupsuch the OR² or OR³ is an ester of an amino acid, such as an ester ofglycine or alanine

Pharmaceutically acceptable salts of these compounds as well aspharmaceutical formulation of these compounds are also provided.

Generally, the compounds described herein have enhanced oralbioavailability compared to the oral bioavailability of treprostinil,either in free acid or salt form. The described compounds can have oralbioavailability that is at least 25%, 50% 100%, 200%, 400% or morecompared to the oral bioavailability of treprostinil. The absolute oralbioavailability of these compounds can range between 10%, 15%, 20%, 25%,30% and 40%, 45%, 50%, 55%, 60% or more when administered orally. Forcomparison, the absolute oral bioavailability of treprostinil is on theorder of 10%, although treprostinil sodium has an absolutebioavailability approximating 100% when administered by subcutaneousinfusion.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein, and in particular the bioavailability rangesdescribed herein also encompass any and all possible subranges andcombinations of subranges thereof. As only one example, a range of 20%to 40%, can be broken down into ranges of 20% to 32.5% and 32.5% to 40%,20% to 27.5% and 27.5% to 40%, etc. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

Administration of these compounds can be by any route by which thecompound will be bioavailable in effective amounts including oral andparenteral routes. The compounds can be administered intravenously,topically, subcutaneously, intranasally, rectally, intramuscularly,transdermally or by other parenteral routes. When administered orally,the compounds can be administered in any convenient dosage formincluding, for example, capsule, tablet, liquid, suspension, and thelike.

Testing has shown that that treprostinil can be irritating upon skincontact. In contrast, some of the compounds disclosed herein, generallyas prodrugs of treprostinil, are not irritating to the skin.Accordingly, the present compounds are well suited for topical ortransdermal administration.

When administered to a subject, the above compounds, and in particularthe compounds of structure I, are prostacyclin-mimetic and are useful intreating conditions or disorders where vasodilation and/or inhibition ofplatelet aggregation or other disorders where prostacyclin has shownbenefit, such as in treating pulmonary hypertension. Accordingly, thepresent invention provides methods for inducing prostacyclin-likeeffects in a subject comprising administering a pharmaceuticallyeffective amount of one or more of the compounds described herein, suchas those of structure I above, preferably orally, to a patient in needof such treatment. As an example, the vasodilating effects of thepresent compounds can be used to treat pulmonary hypertension, whichresult from various forms of connective tissue disease, such as lupus,scleroderma or mixed connective tissue disease. These compounds are thususeful for the treatment of pulmonary hypertension.

In another embodiment, the present invention also provides methods ofpromoting prostacyclin-like effect in a subject by administering apharmaceutically effective amount of a compound of structure II:

wherein,

R¹ is independently selected from the group consisting of H, substitutedand unsubstituted alkyl groups, arylalkyl groups and groups wherein OR¹form a substituted or unsubstituted glycolamide ester;

R² and R³ may be the same or different and are independently selectedfrom the group consisting of H, phosphate and groups wherein OR² and OR³form esters of amino acids or proteins, with the proviso that all of R¹,R² and R³ are not H;

an enantiomer of the compound; and

a pharmaceutically acceptable salt of the compound.

In groups wherein OR¹ form a substituted or unsubstituted glycolamideester, R¹ can be —CH₂CONR⁴R⁵, wherein R⁴ and R⁵ may be the same ordifferent and are independently selected from the group consisting of H,OH, substituted and unsubstituted alkyl groups, —(CH₂)_(m)CH₃, —CH₂OH,and —CH₂(CH₂)_(n)H, with the proviso that m is 0, 1, 2, 3 or 4, and n is0, 1, 2, 3 or 4.

In other methods of inducing vasodilation or treating hypertension, R¹can be a C₁-C₄ alkyl group, such as methyl, ethyl, propyl or butyl. Inother methods R¹ is a substituted or unsubstituted benzyl groups, suchas —CH₂C₆H₅, —CH₂C₆H₄NO₂, —CH₂C₆H₄OCH₃, —CH₂C₆H₄Cl, —CH₂C₆H₄(NO₂)₂, or—CH₂C₆H₄F. The benzyl group can be ortho, meta, para, ortho/parasubstituted and combinations thereof. Suitable substituents on thearomatic ring include halogens (fluorine, chlorine, bromine, iodine),—NO₂ groups, —OR¹⁶ groups wherein R¹⁶ is H or a C₁-C₄ alkyl group, andcombinations thereof.

Alternatively, when R¹ is —CH₂CONR⁴R⁵ then R⁴ and R⁵ may be the same ordifferent and are independently selected from the group consisting of H,OH, —CH₃, and —CH₂CH₂OH. In these methods, where R¹ is not H, generallyone or both of R² and R³ are H.

In some methods, one or both of R² and R³ are H and R¹ is —CH₃,—CH₂C₆H₅. In other methods where one or both of R² and R³ are H, then R¹is —CH₂CONR⁴R⁵, and one or both of R⁴ and R⁵ are H, —OH, —CH₃,—CH₂CH₂OH.

In methods where one or both of R² and R³ are not H, R² and R³ can beindependently selected from phosphate and groups wherein OR² and OR³ areesters of amino acids, dipeptides, esters of tripeptides and esters oftetrapeptides. In some embodiments, only one of R² or R³ is a phosphategroup. In methods where at least one of R² and R³ is not H, generally R¹is H. In other methods, one of R² or R³ is H and the other R² or R³ isas defined elsewhere herein. In some methods, R² is H and R³ is not H.In additional embodiments, R¹ and R³ are H and R² is a group wherein OR²is an ester of an amino acid or a dipeptide. In further embodiments, R¹and R² are H and R³ is a group wherein OR³ is an ester of an amino acidor a dipeptide.

In the methods, where one or both of the OR² and OR³ groups form estersof amino acids or peptides, i.e., dipeptides, tripeptides ortetrapeptides, these can be depicted generically as —COCHR⁶NR⁷R⁸ whereinR⁶ is selected from the group consisting of amino acid side chains, R⁷and R⁸ may be the same or different and are independently selected fromthe group consisting of H, and —COCHR⁹NR¹⁰R¹¹. In the embodimentswherein the amino acid is proline, R⁷ together with R⁶ forms apyrrolidine ring structure. R⁶ can be any of the naturally occurringamino acid side chains, for example —CH₃ (alanine), —(CH₂)₃NHCNH₂NH(arginine), —CH₂CONH₂ (asparagine), —CH₂COOH (aspartic acid), —CH₂SH(cysteine), —(CH₂)₂CONH₂ (glutamine), —(CH₂)₂COOH (glutamic acid), —H(glycine), —CHCH₃CH₂CH₃ (isoleucine), —CH₂CH(CH₃)₂ (leucine), —(CH₂)₄NH₂(lysine), —(CH₂)₂SCH₃ (methionine), —CH₂Ph (phenylalanine), —CH₂OH(serine), —CHOHCH₃ (threonine), —CH(CH₃)₂ (valine),

—(CH₂)₃NHCONH₂ (citrulline) or —(CH₂)₃NH₂ (ornithine). Ph designates aphenyl group.

In the above methods, R⁷ and R⁸ may be the same or different and areselected from the group consisting of H, and —COCHR⁹NR¹⁰R¹¹, wherein R⁹is a side chain of amino acid, R¹⁰ and R¹¹ may be the same or differentand are selected from the group consisting of H, and —COCHR¹²NR¹³R¹⁴,wherein R¹² is an amino acid side chain, R¹³ and R¹⁴ may be the same ordifferent and are independently selected from the group consisting of H,and —COCHR¹⁵NH₂. One skilled in the art will realize that the peptidechains can be extended on the following scheme to the desired length andinclude the desired amino acid residues.

In the embodiments where either or both of OR² and OR³ groups form anester of a peptide, such as dipeptide, tripeptide, tetrapeptide, etc.the peptides can be either homopeptides, i.e., repeats of the same aminoresidue, or heteropeptides, i.e., made up of different combinations ofamino acids.

As will be understood by the skilled artisan when only one of R⁷ and R⁸includes a peptide bond to further amino acid, such as in the di, triand tetrapeptides, the resulting peptide chain will be linear. When bothR⁷ and R⁸ include a peptide bond, then the peptide can be branched.

In still other methods R¹ is H and one of R² or R³ is a phosphate groupor H while the other R² or R³ is a group such the OR² or OR³ is an esterof an amino acid, such as an ester of glycine or alanine

In some methods, the administered compound can have an oralbioavailability that is at least 25%, 50% 100%, 200%, 400% of the oralbioavailability of treprostinil. It is generally preferred to administercompounds that have higher absolute oral bioavailabilities, such as 15%,20%, 25%, 30% and 40%, 45%, 50%, 55%, 60% or more when administeredorally.

Treprostinil has also been discovered to inhibit metastasis of cancercells as disclosed in U.S. patent application Ser. No. 10/006,197 filedDec. 10, 2001 and Ser. No. 10/047,802 filed Jan. 16, 2002, both of whichare hereby incorporated into this application. Accordingly, thecompounds described above, and in particular those of structure I andII, can also be used in the treatment of cancer and cancer relateddisorders, and as such the present invention provides pharmaceuticalcompositions and methods for treating cancer. Suitable formulations andmethods of using the present compounds can be achieved by substitutingthe compounds of the present invention, such as those of structure I andII and in particular prodrugs of treprostinil, for the active compoundsdisclosed in U.S. patent application Ser. Nos. 10/006,197 and 10/047,802filed Jan. 16, 2002.

Synthesis of the following compounds of structure I and structure II canbe achieved as follows:

Synthesis of methyl ester of Treprostinil (2) and biphosphate ester ofTreprostinil

Synthesis of methyl ester of Treprostinil (2)

Methyl ester of treprostinil (2) was prepared by treating 1.087 g (2.8mmoles) of treprostinil (1) with 50 ml of a saturated solution of dryhydrochloric acid in methanol. After 24 hours at room temperature, themethanol was evaporated to dryness and the residue was taken in 200 mldichloromethane. The dichloromethane solution was washed with a 10%aqueous potassium carbonate solution, and then with water to a neutralpH, it was dried over sodium sulfate, filtered and the solvent wasremoved in vacuo affording treprostinil methyl ester (2) in 98% yield asa yellow oil. The crude methyl ester was used as such in subsequentreactions.

Synthesis of biphosphate ester of Treprostinil (4)

The procedure was adapted after Steroids, 2(6), 567-603 (1963). Themethyl ester of treprostinil (2) (60 mg, 0.15 mmoles) was dissolved in 2ml dry pyridine and a pyridinium solution of the previously preparedpyridinium solution of 2-cyanoethylphosphate 1M (0.3 ml, 0.3 mmoles)(cf. Methods in Enzymology, 1971, 18(c), 54-57) were concentrated todryness in vacuo at 40° C. Anhydrous pyridine was added and the reactionmixture was again concentrated; the operation was repeated twice inorder to remove water completely. Finally the residue was dissolved in 2ml anhydrous pyridine and 190 mg (0.9 mmoles) dicyclohexylcarbodiimidewere added as a solution in 2 ml anhydrous pyridine. The reactionmixture in a closed flask was stirred magnetically for 48 hours at roomtemperature. 1 ml water was added and after one hour, the mixture wasconcentrated to a thick paste in vacuo. The reaction mixture was treatedovernight at room temperature with 3 ml of a 1/9 water/methanol solutioncontaining 35 mg sodium hydroxide. The white solid (dicyclohexylurea)formed was removed by filtration and it was washed well with water. Theaqueous-methanolic solution was concentrated almost to dryness in vacuo,water was added and the solution was extracted with n-butanol (3×2 ml),then with methylene chloride (1×2 ml). The pH of the solution wasadjusted to 9.0 by treatment with a sulfonic acid ion exchange resin (H+cycle—Dowex), treatment with Dowex resin for a longer time (˜12 hours)lead to both the cleavage of the TBDMS group and the recovery of thefree carboxyl group. The resin was filtered and the solution wasconcentrated to dryness affording the corresponding bisphosphate 4 (43mg, yield 52%).

Synthesis of 3′-monophosphate ester of treprostinil (8) and2-monophosphate ester of treprostinil (10)

Synthesis of Monoprotected TBDMS methyl ester of treprostinil (5 and 6)

The procedure was adapted from Org. Synth., 1998, 75, 139-145. Thetreprostinil methyl ester (2) (305.8 mg, 0.75 mmoles) was dissolved in15 ml anhydrous dichloromethane and the solution was cooled on an icebath to 0° C. Imidazole (102 mg, 1.5 mmoles) and tert-butyldimethylsilyl chloride (226.2 mg, 1.5 mmoles) were added and the mixture wasmaintained under stirring at 0° C. for 30 minutes, then stirredovernight at room temperature. Water (25 ml) was added and the organiclayer was separated. The aqueous layer was then extracted withdichloromethane (3×50 ml). The organic layers were dried over Na₂SO₄,the solution was filtered and the solvent was removed in vacuo affording447 mg crude reaction product. The crude reaction product was separatedby column chromatography (silica gel, 35% ethyl acetate/hexanes)affording 140 mg bis-TBDMS protected Treprostinil methyl ester, 160 mg2-TBDMS protected treprostinil methyl ester (6) and 60 mg 3′-TBDMSprotected Treprostinil methyl ester (5).

Synthesis of monophosphate ester of Treprostinil 8/10

The procedure was adapted after Steroids, 1963, 2(6), 567-603 and is thesame for (8) and (10) starting from (6) and (5), respectively. The TBDMSprotected methyl ester of treprostinil (6) (46 mg, 0.09 mmoles) wasdissolved in 2 ml dry pyridine and a pyridinium solution of thepreviously prepared pyridinium solution of 2-cyanoethylphosphate 1M (0.2ml, 0.2 mmoles) (cf. Methods in Enzymology, 1971, 18(c), 54-57) wereconcentrated to dryness in vacuo at 40° C. Anhydrous pyridine was addedand the reaction mixture was again concentrated; the operation wasrepeated twice in order to remove water completely. Finally the residuewas dissolved in 2 ml anhydrous pyridine and 116 mg (0.56 mmoles)dicyclohexylcarbodiimide were added as a solution in 2 ml anhydrouspyridine. The reaction mixture in a closed flask was stirredmagnetically for 48 hours at room temperature in the dark. 5 ml waterwere added and after one hour, the mixture was concentrated to a thickpaste in vacuo. The reaction mixture was treated overnight at roomtemperature with 10 ml of a 1/9 water/methanol solution containing 100mg sodium hydroxide. The white solid (dicyclohexylurea) formed wasremoved by filtration and it was washed well with water. Theaqueous-methanolic solution was concentrated almost to dryness in vacuo,water was added and the solution was extracted with n-butanol (3×10 ml),then with methylene chloride (1×10 ml). The pH of the solution wasadjusted to 9.0 by treatment with a sulfonic acid ion exchange resin (H+cycle—Dowex); treatment with Dowex resin for a longer time (˜12 hours)lead to both the cleavage of the TBDMS group and the recovery of thefree carboxyl group. The resin was filtered and the solution wasconcentrated to dryness affording the corresponding monophosphate 8 (33mg, yield 68%).

Synthesis of methyl ester of treprostinil (2)

(2) (1 g; 2.56 mmol) was added to methanol (50 ml) prior saturated withgaseous hydrochloric acid and the mixture swirled to give a clearsolution that was left to stand overnight at room temperature. Solventwas removed in vacuo and the residue was neutralized with a 20%potassium carbonate solution and extracted in dichloromethane. Theorganic layer was washed with water, dried over anhydrous magnesiumsulfate and evaporated to yield the crude product (0.96 g). Purificationby preparative tlc (silica gel plate; eluent: 7:3 (v/v) hexane-ethylacetate) afforded 2 (0.803; 77.5%), colorless oil.

Synthesis of Tritreprostinil diethanolamine (UT-15C)

Treprostinil acid is dissolved in a 1:1 molar ratio mixture ofethanol:water and diethanolamine is added and dissolved. The solution isheated and acetone is added as an antisolvent during cooling.

Synthesis of diglycil ester of treprostinil methyl ester (12)

To a magnetically stirred solution of (2) methyl ester 2 (0.268 g; 0.66mmol) in dichloromethane (30 ml) N-carbobenzyloxyglycine p-nitrophenylester (0.766 g; 2.32 mmol) and 4-(dimethyamino)pyridine (250 mg; 2.05mmol) were successively added. The resulted yellow solution was stirredat 20° C. for 24 hrs., then treated with 5% sodium hydroxide solution(20 ml) and stirring continued for 15 mm. Dichloromethane (50 ml) wasadded, layers separated and the organic phase washed with a 5% sodiumhydroxide solution (6×20 ml), water (30 ml), 10% hydrochloric acid (2×40ml), 5% sodium bicarbonate solution (40 ml) and dried over anhydroussodium sulfate. Removal of the solvent afforded crude (11) (0.61 g),pale-yellow viscous oil. Purification by flash column chromatography onsilica gel eluting with gradient 9/1 to 1/2 (v/v) hexane-ethyl etherafforded 0.445 g (85.3%) of 11, white crystals, m.p. 70-72° C. ′Fl-NMR[CDCl₃; δ(ppm)]: 3.786 (s) (3H, COOCH ₃), 3.875 (d) (2H) and 3.940 (d)(2H) (NH—CH ₂—COO), 4.631 (s) (2H, OCH ₂COOCH₃), 4.789 (m)(1H, adjacentto OOC—CH₂NHcbz) and 4.903 (m) (1H, adjacent to OOCCH₂NHcbz), 5.09(s)(4H, C₆H₅CH ₂O), 5.378 (m)(1H) and 5.392 (m)(1H)(NH), 7.295-7.329(m)(10H, C₆H₅). LR ESI-MS (m/z): 787.1 [M+H]⁺, 804.1 [M+NH₄]⁺, 809.3[M+Na]⁺, 825.2 [M+K]⁺, 1590.5 [2M+NH₄]⁺, 1595.6 [2M+Na]₊.

Methyl ester, diglycyl ester (12)

A solution of ester (11) (0.4 g; 0.51 mmol) in methanol (30 ml) wasintroduced in the pressure bottle of a Parr hydrogenation apparatus, 10%palladium on charcoal (0.2 g; 0.197 mmol Pd) was added, apparatusclosed, purged thrice with hydrogen and loaded with hydrogen at 50p.s.i. Stirring was started and hydrogenation carried out for 5 hrs. atroom temperature. Hydrogen as removed from the installation by vacuumsuction and replaced with argon. The catalyst was filtered off throughcelite deposited on a fit and the filtrate concentrated in vacuo to give0.240 g (91%) of 4, white solid m.p. 98-100° C.

Synthesis of benzyl ester of treprostinil (13)

To a stirred solution of (2) (2 g; 5.12 mmol) in anhydroustetrahydrofuran (20 ml) benzyl bromide (0.95 ml; 7.98 mmol) and freshlydistilled triethylamine (1.6 ml; 11.48 mmol) were consecutively added atroom temperature and the obtained solution was refluxed with stirringfor 12 hrs. A white precipitate was gradually formed. Solvent wasdistilled off in vacuo and the residue treated with water (30 ml). Uponextraction with methylene chloride emulsion formation occurs. Theorganic and aqueous layers could be separated only after treatment with5% hydrochloric acid solution (20 ml). The organic layer was washed withwater, dried on anhydrous sodium sulfate, and evaporated, the residuewas further dried under reduced pressure over phosphorus pentoxide togive a yellow viscous oil (2.32 g) that was purified by preparative thinlayer chromatography (silica gel plate; eluent: 1:2, v/v, hexane/ethylether). Yield: 81.2%.

Synthesis of bis-glycyl ester of treprostinil (15)

Benzy ester, di-cbzGly ester (14)

To a magnetically stirred solution of benzyl ester 13 (1 g; 2.08 mmol)in dichloromethane (50 ml) N-carbobenzyloxyglycine p-nitrophenyl ester(2.41 g; 7.28 mmol) and 4-(dimethyamino) pyridine (788 mg; 6.45 mmol)were added. The resulted yellow solution was stirred at 20° C. for 21hrs., then successively washed with a 5% sodium hydroxide solution (6×45ml), 10% hydrochloric acid (2×40 ml), 5% sodium bicarbonate solution (40ml) and dried over anhydrous sodium sulfate. Removal of the solvent,followed by drying over phosphorus pentoxide under reduced pressure,afforded crude 14 (2.61 g), pale-yellow oil. Purification by flashcolumn chromatography on silica gel eluting with gradient 9:1 to 1:2(v/v) hexane-ethyl ether gave (14 (1.51 g; 84.1%) as a colorless, veryviscous oil.

Diglycyl ester (15)

A solution of ester (14) (0.4 g; 0.46 mmol) in methanol (30 ml) washydrogenated over 10% Pd/C as described for ester (12). Work-up anddrying over phosphorus pentoxide in vacuo yielded 0.170 g (72.7%) ofester 15, white solid m.p. 155-158° C.

Synthesis of 3′-glycyl ester of treprostinil 19

Benzyl ester, t-butyldimethysilyl monoester (16)

A solution of tert-butyldimethylsilyl chloride (0.45 g; 2.98 mmol) indichloromethane (8 ml) was added dropwise over 10 min., at roomtemperature, into a stirred solution of benzyl ester 13 (0.83 g; 1.73mmol) and imidazole (0.33 g; 4.85 mmol) in dichloromethane (20 ml).Stirring was continued overnight then water (20 ml) was added, themixture stirred for one hour, layers separated, organic layer dried overanhydrous sodium sulfate and concentrated in vacuo to give a slightlyyellow oil (1.15 g). The crude product is a mixture of the mono-TBDMS(16) and di-TBDMS esters (¹H-NMR). Column chromatography on silica gel,eluting with a 9:1 (v/v) hexane-ethyl acetate mixture, readily affordedthe di-ester (0.618 g) in a first fraction, and ester 16 (0.353 g; yieldrelative to 13: 34.4%) in subsequent fractions. Analytical tlc on silicagel of the ester 16 showed only one spot (eluent: 3:2 (v/v) hexane-ethylether). Consequently, under the above reaction conditions, the otherpossible isomer (mono-TBDMS ester at the side-chain hydroxyl) was notobserved.

Another experiment in which the molar ratio tert-butyldimethylsilylchloride:ester 13 was lowered to 1.49 (followed by flash columnchromatography of the product on silica gel, eluting with gradient9.5/0.5 to 3/1 (v/v) hexane-ethyl ether) lead to a decreased content(36.5%, as pure isolated material) of the undesired di-OTBDMSby-product. The mono-OTBDMS ester fractions (45.1%; isolated material)consisted of ester 16 (98%) and its side-chain isomer (2%) that could bedistinctly separated; the latter was evidenced (tic, NMR) only in thelast of the monoester fractions.

Benzyl ester, cbz-glycyl monoester (18)

To a magnetically stirred solution of ester 16 (0.340 g; 0.57 mmol) indichloromethane (15 ml) N-carbobenzyloxyglycine p-nitrophenyl ester(0.445 g; 1.35 mmol) and 4-(dimethyamino) pyridine (150 mg; 1.23 mmol)were successively added. The solution was stirred at 20° C. for 40 hrs.Work-up as described for esters 11 and 14 yielded a crude product (0.63g) containing 90% 17 and 10% 18 (¹H-NMR). To completely remove theprotective TBDMS group, this mixture was dissolved in ethanol (30 ml)and subjected to acid hydrolysis (5% HCl, 7 ml) by stirring overnight atroom temperature. Solvent was then removed under reduced pressure andthe residue extracted in dichloromethane (3×50 ml); the organic layerwas separated, washed once with water (50 ml), dried over sodium sulfateand concentrated in vacuo to give crude ester 18 (0.51 g). Purificationby flash column chromatography as for esters 11 and 14 afforded ester 18(0.150 g; overall yield: 39.1%) as a colorless, viscous oil.

Glycyl monoester (19)

A solution of ester 18 (0.15 g; 0.22 mmol) in methanol (30 ml) washydrogenated over 10% Pd/C as described for ester 12 and 15. Work-up anddrying over phosphorus pentoxide in vacuo yielded ester 10 (0.98 g;98.0%), white, shiny crystals m.p. 74-76° C. LR ESI-MS (m/z): 448.2[M+H]⁺, 446.4 [M−H]⁻.

Synthesis of 3′-L-leucyl ester of treprostinil 22

Benzyl ester, t-butyldimethysilyl monoester, cbz-L-leucyl monoester (20)

To a stirred solution of ester 16 (0.38 g: 0.64 mmol) andN-carbobenzyloxy-L-leucine N-hydroxysuccinimide ester (0.37 g; 1.02mmol) in 10 ml dichloromethane 4-(dimethyamino)pyridine (0.17 g; 1.39mmol) was added, then stirring continued at room temperature for 2 days.The solvent was removed in vacuo and the crude product (0.9 g) subjectedto flash column chromatography on silica gel eluting with 9:1hexane-ethyl acetate; the firstly collected fraction yielded an oil(0.51 g) which, based on the its NMR spectrum and tlc, was proved to bea 2:1 mixture of ester 20 and the starting ester 16. Preparative tlc onsilica gel (eluent: ethyl acetate-hexane 1:4) gave pure 20, colorlessoil (overall yield based on 7: 62.6%).

Benzyl ester, cbz-L-leucyl monoester (21)

De-protection of the cyclopentenyl hydroxyl in the t-butyldimethysilylmonoester 20 succeeded by treatment with diluted hydrochloric acidsolution as described for 18, with the exception that a 1:5 (v/v)chloroform-ethanol mixture, instead of ethanol alone, was used to ensurehomogeneity. Work-up afforded 20, colorless oil, in 87.6% yield.

L-leucyl monoester (22)

Hydrogenolysis of the benzyl and N-carbobenzyloxy groups in 21 wascarried out as for 18. Work-up afforded 22 (95.3%), white solid, m.p.118-120° C.

Synthesis of 2-L-leucyl ester of treprostinil 25

Benzyl ester, cbz-L-leucyl monoesters (21, 23) and -diester (24)

To a stirred solution of ester 13 (0.53 g: 1.10 mmol) andN-carbobenzyloxy-L-leucine N-hydroxysuccinimide ester (0.76 g; 2.05mmol) in dichloromethane (30 ml) 4-(dimethyamino) pyridine (0.29 g; 2.37mmol) was added, then stirring continued at room temperature for 1 day.The solution was diluted with dichloromethane (40 mnl), successivelywashed with a 5% sodium hydroxide solution (4×25 ml), 10% hydrochloricacid (2×30 ml), 5% sodium bicarbonate solution (50 ml), dried overanhydrous sodium sulfate and concentrated under reduced pressure to givethe crude product (0.85 g), as a viscous, yellow oil. Thin layerchromatography revealed a complex mixture in which esters 13 and 21 aswell as cbz-L-leucine could be identified through the correspondingr_(F) values, only as minor products. The crude product wasflash-chromatographed through a silica gel column eluting with gradienthexane-ethyl ether. At 7:3 (v/v) hexane-ethyl ether, the first fractiongave the cbz-L-leucyl diester 24 (6% of the product subjected tochromatography) while the two subsequent fractions afforded thecbz-L-leucyl monoester 23 (54% of the crude product, as pure isolated23; 57.6% yield, relative to 2). Purity of both compounds was verifiedby analytical tlc and NMR. The other isomer, cbz-L-leucyl monoester 21constituted only about 5% of the crude product and was isolated bypreparative tlc of the latter only a 3:1 23/21 mixture.

L-leucyl monoester (25)

Hydrogenolysis of 23 to the ester 25 was performed as described forcompound 12 but reaction was carried out at 35 p.s.i., overnight.Work-up and drying over phosphorus pentoxide in vacuo afforded 25, whitesolid m. p. 153-155° C., in quantitative yield.

Synthesis of 3′-L-alanyl ester of treprostinil 30

N-Cbz-L-alanyl p-nitro phenyl ester (27)

To a stirred solution containing N-carbobenzyloxy-L-alanine (1 g; 4.48mmol) and p-nitrophenol (1 g; 7.19 mmol) in anhydrous tetrahydrofuran (7ml) a fine suspension of 1,3-dicyclohexylcarbodiimide (1.11 g; 5.38mmol) in tetrahydrofuran (5 ml) was added over 30 min. Stirring wascontinued at room temperature for 18 hrs., glacial acetic acid (0.3 ml)added, 1,3-dicyclohexylurea filtered off and solvent removed in vacuo,at 40° C., to give a viscous, yellow-reddish oil (2.5 g). The ¹H-NMRspectrum showed a mixture consisting of N-carbobenzyloxy-Lalaninep-nitrophenyl ester (27), unreacted p-nitrophenol and a small amount ofDCU, which was used as such in the next reaction step.

Benzyl ester, cbz-L-alanyl monoester (29)

A solution of 4-(dimethylamino)pyridine (0.30 g; 2.49 mmol) indichloromethane (3 ml) was quickly dropped (over 5 min.) into amagnetically stirred solution of ester 16 (0.37 g; 0.62 mmol) and crudeN-carbobenzyloxy-L-alanine p-nitrophenyl ester (0.98 g) indichloromethane (12 ml). The mixture was stirred overnight at roomtemperature, then diluted with dichloromethane (50 ml), and thoroughlywashed with a 5% sodium hydroxide solution (7×35 ml), 10% hydrochloricacid (3×35 ml), 5°/a sodium bicarbonate solution (50 ml), dried overanhydrous sodium sulfate and concentrated under reduced pressure to givethe crude ester 28 (1.1 g). The latter was dissolved in ethanol (30 ml),5% hydrochloric acid (8 ml) and chloroform (5 ml) were added and thesolution stirred overnight. Solvents were removed in vacuo, the residuetaken-up in dichloromethane, washed to pH 7 with a 5% sodiumhydrogencarbonate solution, dried over anhydrous sodium sulfate and thesolvent evaporated affording crude 29 (1.04 g). Purification by columnchromatography on silica gel, eluting with gradient hexane-ethyl ether,enabled separation of a fraction (at hexane:ethyl ether=1:1 v/v) of pure29 as a colorless very viscous oil (0.11 g; 25.8% overall yield, basedon 16).

L-alanyl monoester (30)

Removal of the benzyl and N-carbobenzyloxy groups in 29 was achievedthrough catalytic hydrogenation as described for 12. Ester 30 wasobtained (yield: 97.2%) as a pale-yellow, partially crystallized, oil.

Synthesis of the 3′-L-valine ester of Treprostinil benzyl ester 33

Synthesis of the benzyl ester of Treprostinil 13

The benzyl ester 11 was synthesized by adapting the method described byJ. C. Lee et al. in Organic Prep. and Proc. Intl., 1996, 28(4), 480-483.To a solution of 1 (620 mg, 1.6 mmoles) and cesium carbonate (782.4 mg,2.4 mmoles) in acetonitrile (30 ml) was added benzyl bromide (0.48 ml, 4mmoles) and the mixture was stirred at reflux for 1 hour. After coolingat room temperature, the precipitate was filtered off and the filtratewas concentrated in vacuo. The residue was dissolved in chloroform (150ml) and washed with a 2% aqueous solution of NaHCO₃ (3×30 ml). Theorganic layer was washed with brine, dried on Na₂SO₄, filtered and thesolvent was removed in vacuo to afford 750 mg of the crude benzyl ester13 (yield 98%) as a yellow viscous oil. The crude benzyl ester 13 can bepurified by column chromatography (100-0% dichloromethane(methanol) butit can also be used crude in subsequent reactions.

Synthesis of the TBDMS protected Treprostinil benzyl ester 16

The procedure for the synthesis of the TBDMS protected benzyl ester wasadapted from Organic Synth., 1998, 75, 139-145. The benzyl ester 13 (679mg, 1.4 mmoles) was dissolved in anhydrous dichloromethanc (20 ml) andthe solution was cooled to 0° C. on an ice bath. Imidazole (192 mg, 2.8mmoles) and t-butyl-dimethylsilyl chloride (TBDMSC1) (420 mg, 2.8mmoles) were added and the mixture was maintained under stirring foranother half hour on the ice bath and then it was left overnight at roomtemperature. 40 ml water was added to the reaction mixture and theorganic layer was separated. The aqueous layer was extracted with 3×50ml dichloromethane. The combined organic layers were dried over Na₂SO₄,filtered and the solvent was removed in vacuo. This afforded 795 mg ofmaterial which proved to be a mixture of the desired mono TBDMSprotected 5 benzyl ester with the bis-TBDMS protected benzyl ester. Pure16 (249 mg) was obtained by column chromatography on silica gel (eluent35% ethyl acetate/hexane).

Synthesis of N-Cbz-L-valine ester of the TBDMS protected Treprostinilbenzyl ester 31

The procedure used was adapted from Tetrahedron Lett., 1978, 46,4475-4478. A solution of NCbz-L-valine (127 mg, 0.5 mmoles),N,N-dicyclohexylcarbodiimide (DCC) (111 mg, 0.5 mmoles), compound 16(249 mg, 0.4 mmoles) and 4-(dimethylamino)pyridine (DMAP) (6 mg, 0.05mmoles) in anhydrous dichloromethane (15 ml) was stirred at roomtemperature until esterification was complete. The solution was filteredand the formed N,N-dicyclohexylurea was filtered. The filtrate wasdiluted with dichloromethane (80 ml) and washed with water (3×30 ml), a5% aqueous acetic acid solution (2×30 ml) and then again with water(3×30 ml). The organic layer was dried over Na₂SO₄ and the solvent wasevaporated in vacuo affording 369 mg crude 31. Pure 31 was obtained bychromatography (silica gel, 35% ethyl acetate/hexane).

Synthesis of the 3′-N-Cbz-L-valine ester of Treprostinil benzyl ester 32

Cleavage of the TBDMS group in compound 31 was achieved using anadaptation of the procedure described in Org. Letters, 2000, 2(26),4177-4180. The N-Cbz-L-valine ester of the TBDMS protected benzyl ester31 (33 mg, 0.04 mmoles) was dissolved in methanol (5 ml) andtetrabutylammonium tribromide (TBATB) (2 mg, 0.004 mmoles) was added.The reaction mixture was stirred at room temperature for 24 hrs untilthe TBDMS deprotection was complete. The methanol was evaporated and theresidue was taken in dichloromethane. The dichloromethane solution waswashed with brine and then dried over Na₂SO₄. After filtering the dryingagent the solvent was evaporated to dryness affording 30.2 mg of crudecompound 32.

Synthesis of the 3′-L-valinc ester of Treprostinil 33

The benzyl and benzyl carboxy groups were removed by catalytichydrogenation at atmospheric pressure in the presence of palladium 10%wt on activated carbon. The 3′-N-Cbz-L-valine ester of benzyl ester 32(30.2 mg, 0.04 mmoles) was dissolved in methanol (10 ml) and a catalyticamount of Pd/C was added. Under magnetic stirring the air was removedfrom the flask and then hydrogen was admitted. The reaction mixture wasmaintained under hydrogen and stirring at room temperature for 24 hrs,then the hydrogen was removed with vacuum. The reaction mixture was thenfiltered through a layer of celite and the solvent was removed in vacuoto afford the pure 3′-L-valine ester of Treprostinil 33 (15 mg, 0.03mmoles).

Synthesis of 2-L-valine ester of Treprostinil 36/bis-L-valine ester ofTrenrostinil 37 Synthesis of 2-L-alanine ester of Treprostinil36′/bis-L-alanine ester of Treprostinil 37′

Synthesis of 2-N-Cbz-L-valine ester of Treprostinil benzyl ester 34 andbis-N-Cbz-L-valine ester of Treprostinil benzyl ester 35

The procedure used was adapted from Tetrahedron Lett., 1978, 46,4475-4478. A solution of NCbz-L-valine (186 mg, 0.7 mmoles),N,N-dicyclohexylcarbodiimide (DCC) (167 mg, 0.8 mmoles), compound 13(367 mg, 0.8 mmoles) and 4-(dimethylamino)pyridine (DMAP) (12 mg, 0.09mmoles) in anhydrous dichloromethane (15 ml) was stirred at roomtemperature until esterification was complete. The solution was filteredand the formed N,N-dicyclohexylurea was filtered. The filtrate wasdiluted with dichloromethane (100 ml) and washed with water (3×50 ml), a5% aqueous acetic acid solution (2×50 ml) and then again with water(3×50 ml). The organic layer was dried over Na₂SO₄ and the solvent wasevaporated in vacuo affording 556 mg crude product. The product wasseparated by chromatography (silica gel, 35% ethyl acetate/hexane)yielding 369.4 mg 2-valine ester 34 and 98 mg bis-valine ester 35.

Synthesis of 2 N-Cbz-L-alanine ester of Treprostinil benzyl ester 34′and bis-N-Cbz-L-alanine ester of Treprostinil benzyl ester 35′

The procedure used was adapted from Tetrahedron Lett., 1978, 46,4475-4478. A solution of NCbz-L-alanine (187 mg, 0.84 mmoles),N,N-dicyclohexylcarbodiimide (DCC) (175 mg, 0.85 mmoles), compound 13(401 mg, 0.84 mmoles) and 4-(dimethylamino)pyridine (UMAP) (11.8 mg, 0.1mmoles) in anhydrous dichloromethane (15 ml) was stirred at roomtemperature until esterification was complete. The solution was filteredand the formed N,N-dicyclohexylurea was filtered. The filtrate wasdiluted with dichloromethane (100 ml) and washed with water (3×50 ml), a5% aqueous acetic acid solution (2×50 ml) and then again with water(3×50 ml). The organic layer was dried over Na₂SO₄ and the solvent wasevaporated in vacuo affording 516 mg crude product. The product wasseparated by chromatography (silica gel, 35% ethyl acetate/hexane)yielding 93.4 mg 2-alanine ester 34′ and 227 mg bis-alanine ester 35′.

Synthesis of 2-L-valine ester of Treprostinil 36/bis-L-valine ester ofTreprostinil 37

The benzyl and benzyl carboxy groups were removed by catalytichydrogenation at atmospheric pressure in the presence of palladium 10%wt on activated carbon. The 2-N-Cbz-L-valine ester of Treprostinilbenzyl ester 34 (58.2 mg, 0.08 mmoles)/bis-N-Cbz-L-valine ester ofTreprostinil benzyl ester 35 (55.1 mg, 0.06 mmoles) was dissolved inmethanol (10 ml) and a catalytic amount of Pd/C was added. Undermagnetic stirring the air was removed from the flask and hydrogen wasadmitted. The reaction mixture was maintained under hydrogen andstirring at room temperature for 20 hrs, then hydrogen was removed withvacuum. The reaction mixture was then filtered through a layer of celiteand the solvent was removed in vacuo to afford the pure 2-L-valine esterof Treprostinil 36 (40 mg, 0.078 mmoles)/bis-L-valine ester ofTreprostinil 37 (23 mg, 0.04 mmoles).

Synthesis of 2-L-alanine ester of Treprostinil 36′/bis-L-alanine esterof Treprostinil 37′

The benzyl and benzyl carboxy groups were removed by catalytichydrogenation at atmospheric pressure in the presence of palladium 10%wt on activated carbon. The 2-N-Cbz-L-alanine ester of Treprostinilbenzyl ester 34′ (87.4 mg, 0.13 mmoles)/bis-N-Cbz-L-alanine ester ofTreprostinil benzyl ester 35′ (135 mg, 0.15 mmoles) was dissolved inmethanol (15 ml) and a catalytic amount of Pd/C was added. Undermagnetic stirring the air was removed from the flask and hydrogen wasadmitted. The reaction mixture was maintained under hydrogen andstirring at room temperature for 20 hrs, then hydrogen was removed withvacuum. The reaction mixture was then filtered through a layer of celiteand the solvent was removed in vacuo to afford the pure 2-L-valine esterof Treprostinil 36′ (57 mg, 0.12 mmoles)/bis-L-alanine ester ofTreprostinil 37′ (82 mg, 0.15 mmoles).

Synthesis of benzyl esters of treprostinil 38 a-e

a 4-NO₂C₆H₄CH₂; b 4-(CH₃O)C₆H₄CH₂; c 2-ClC₆H₄CH₂; d 2,4-(NO₂)₂C₆H₃CH₂; e4-FC₆H₄CH₂ Synthesis of the benzyl esters of treprostinil 38 a-e wasperformed using the procedure for the benzyl ester 13.

Enantiomers of these compounds, shown below, can be synthesized usingreagents and synthons of enantiomeric chirality of the above reagents.

(−)-treprostinil can be synthesized as follows:

Briefly, the enantiomer of the commercial drug (+)-Treprostinil wassynthesized using the stereoselective intramolecular Pauson Khandreaction as a key step and Mitsunobu inversion of the side-chainhydroxyl group. The absolute configuration of (−)-Treprostinil wasconfirmed by an X-ray structure of the L-valine amide derivative.

The following procedure was used to make(−)-treprostinil-methyl-L-valine amide: To a stirred solution of(−)-Treprostinil (391 mg, 1 mmol) and L-valine methyl esterhydrochloride (184 mg, 1.1 mmol) in DMF (10 ml) under Ar wassequentially added pyBOP reagent (1.04 g, 2 mmol), diisopropylethylamine (0.52 ml, 3 mmol). The reaction mixture was stirred at roomtemperature overnight (15 hrs). Removal of the solvent in vacuo andpurification by chromatography yielded white solid 12 (481 mg, 86%),which was recrystallized (10% ethyl acetate in hexane) to give suitablecrystals for X-ray.

Various modifications of these synthetic schemes capable of producingadditional compounds discussed herein will be readily apparent to oneskilled in the art.

There are two major barriers to deliver treprostinil in the circulatorysystem. One of these barriers is that treprostinil undergoes a largefirst pass effect. Upon first circulating through the liver, about 60%of treprostinil plasma levels are metabolized, which leaves only about40% of the absorbed dose. Also, a major barrier to oral delivery fortreprostinil is that the compound is susceptible to an efflux mechanismin the gastrointestinal tract. The permeability of treprostinil has beenmeasured across Caco-2 cell monolayers. The apical to basal transportrate was measured to be 1.39×10⁶ cm/sec, which is indicative of a highlypermeable compound. However, the basal to apical transport rate was12.3×10⁶ cm/sec, which suggests that treprostinil is efficientlyeffluxed from the serosal to lumenal side of the epithelial cell. Thesedata suggest that treprostinil is susceptible to p-glycoprotein, amembrane bound multidrug transporter. It is believed that thep-glycoprotein efflux pump prevents certain pharmaceutical compoundsfrom traversing the mucosal cells of the small intestine and, therefore,from being absorbed into systemic circulation.

Accordingly, the present invention provides pharmaceutical compositionscomprising treprostinil, the compound of structure I or the compound ofstructure II, or their pharmaceutically acceptable salts andcombinations thereof in combination with one or more inhibitors ofp-glycoprotein. A number of known non-cytotoxic pharmacological agentshave been shown to inhibit p-glycoprotein are disclosed in U.S. Pat.Nos. 6,451,815, 6,469,022, and 6,171,786.

P-glycoprotein inhibitors include water soluble forms of vitamin E,polyethylene glycol, poloxamers including Pluronic F-68, polyethyleneoxide, polyoxyethylene castor oil derivatives including Cremophor EL andCremophor RH 40, Chrysin, (+)-Taxifolin, Naringenin, Diosmin, Quercetin,cyclosporin A (also known as cyclosporine), verapamil, tamoxifen,quinidine, phenothiazines, and9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7,-dimethoxy-2-isoquinolinyl)ethyl]phenyl]-4-acridinecarboxamideor a salt thereof.

Polyethylene glycols (PEGs) are liquid and solid polymers of the generalformula H(OCH₂CH₂)_(n)OH, where n is greater than or equal to 4, havingvarious average molecular weights ranging from about 200 to about20,000. PEGs are also known asalpha-hydro-omega-hydroxypoly-(oxy-1,2-ethanediyl)polyethylene glycols.For example, PEG 200 is a polyethylene glycol wherein the average valueof n is 4 and the average molecular weight is from about 190 to about210. PEG 400 is a polyethylene glycol wherein the average value of n isbetween 8.2 and 9.1 and the average molecular weight is from about 380to about 420. Likewise, PEG 600, PEG 1500 and PEG 4000 have averagevalues of n of 12.5-13.9, 29-36 and 68-84, respectively, and averagemolecular weights of 570-630, 1300-1600 and 3000-3700, respectively, andPEG 1000, PEG 6000 and PEG 8000 have average molecular weights of950-1050, 5400-6600, and 7000-9000, respectively. Polyethylene glycolsof varying average molecular weight of from 200 to 20000 are well knownand appreciated in the art of pharmaceutical science and are readilyavailable.

The preferred polyethylene glycols for use in the instant invention arepolyethylene glycols having an average molecular weight of from about200 to about 20,000. The more preferred polyethylene glycols have anaverage molecular weight of from about 200 to about 8000. Morespecifically, the more preferred polyethylene glycols for use in thepresent invention are PEG 200, PEG 400, PEG 600, PEG 1000, PEG 1450, PEG1500, PEG 4000, PEG 4600, and PEG 8000. The most preferred polyethyleneglycols for use in the instant invention is PEG 400, PEG 1000, PEG 1450,PEG 4600 and PEG 8000.

Polysorbate 80 is an oleate ester of sorbitol and its anhydridescopolymerized with approximately 20 moles of ethylene oxide for eachmole of sorbitol and sorbitol anhydrides. Polysorbate 80 is made up ofsorbitan mono-9-octadecanoate poly(oxy-1,2-ethandiyl) derivatives.Polysorbate 80, also known as Tween 80, is well known and appreciated inthe pharmaceutical arts and is readily available.

Water-soluble vitamin E, also known as d-alpha-tocopheryl polyethyleneglycol 1000 succinate [TPGS], is a water-soluble derivative ofnatural-source vitamin E. TPGS may be prepared by the esterification ofthe acid group of crystalline d-alpha-tocopheryl acid succinate bypolyethylene glycol 1000. This product is well known and appreciated inthe pharmaceutical arts and is readily available. For example, awater-soluble vitamin E product is available commercially from EastmanCorporation as Vitamin E TPGS.

Naringenin is the bioflavonoid compound2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one andis also known as 4′,5,7-trihydroxyflavanone. Naringenin is the agluconof naringen which is a natural product found in the fruit and rind ofgrapefruit. Naringenin is readily available to the public fromcommercial sources.

Quercetin is the bioflavonoid compound2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one and isalso known as 3,3′,4′,5,7-pentahydroxyflavone. Quercetin is the agluconof quercitrin, of rutin and of other glycosides. Quercetin is readilyavailable to the public from commercial sources.

Diosmin is the naturally occurring flavonic glycoside compound7-[[6-O-6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one.Diosmin can be isolated from various plant sources including citrusfruits. Diosmin is readily available to the public from commercialsources.

Chrysin is the naturally occurring compound5,7-dihydroxy-2-phenyl-4H-1-benzopyran-4-one which can be isolated fromvarious plant sources. Chrysin is readily available to the public fromcommercial sources.

Poloxamers are alpha-hydro-omega-hydroxypoly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers. Poloxamers are aseries of closely related block copolymers of ethylene oxide andpropylene oxide conforming to the general formulaHO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H. For example, poloxamer 124 is aliquid with “a” being 12, “b” being 20, and having an average molecularweight of from about 2090 to about 2360; poloxamer 188 is a solid with“a” being 80, “b” being 27, and having an average molecular weight offrom about 7680 to about 9510; poloxamer 237 is a solid with “a” being64, “b” being 37, and having an average molecular weight of from about6840 to about 8830; poloxamer 338 is a solid with “a” being 141, “b”being 44, and having an average molecular weight of from about 12700 toabout 17400; and poloxamer 407 is a solid with “a” being 101, “b” being56, and having an average molecular weight of from about 9840 to about14600. Poloxamers are well known and appreciated in the pharmaceuticalarts and are readily available commercially. For example, Pluronic F-68is a commercially available poloxamer from BASF Corp. The preferredpoloxamers for use in the present invention are those such as poloxamer188, Pluronic F-68, and the like.

Polyoxyethylene castor oil derivatives are a series of materialsobtained by reacting varying amounts of ethylene oxide with eithercastor oil or hydrogenated castor oil. These polyoxyethylene castor oilderivatives are well known and appreciated in the pharmaceutical artsand several different types of material are commercially available,including the Cremophors available from BASF Corporation.Polyoxyethylene castor oil derivatives are complex mixtures of varioushydrophobic and hydrophilic components. For example, in polyoxyl 35castor oil (also known as Cremophor EL), the hydrophobic constituentscomprise about 83% of the total mixture, the main component beingglycerol polyethylene glycol ricinoleate. Other hydrophobic constituentsinclude fatty acid esters of polyethylene glycol along with someunchanged castor oil. The hydrophilic part of polyoxyl 35 castor oil(17%) consists of polyethylene glycols and glyceryl ethoxylates.

In polyoxyl 40 hydrogenated castor oil (Cremophor RH 40) approximately75% of the components of the mixture are hydrophobic. These comprisemainly fatty acid esters of glycerol polyethylene glycol and fatty acidesters of polyethylene glycol. The hydrophilic portion consists ofpolyethylene glycols and glycerol ethoxylates. The preferredpolyoxyethylene castor oil derivatives for use in the present inventionare polyoxyl 35 castor oil, such as Cremophor EL, and polyoxyl 40hydrogenated castor oil, such as Cremophor RH 40. Cremophor EL andCremophor RH 40 are commercially available from BASF Corporation.

Polyethylene oxide is a nonionic homopolymer of ethylene oxideconforming to the general formula (OCH₂CH₂)_(n) in which n representsthe average number of oxyethylene groups. Polyethylene oxides areavailable in various grades which are well known and appreciated bythose in the pharmaceutical arts and several different types of materialare commercially available. The preferred grade of polyethylene oxide isNF and the like which are commercially available.

(+)-Taxifolin is(2R-trans)-2-(3,4-dihydroxyphenyl)-2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one. Other common names for (+)-taxifolin are(+)-dihydroquercetin; 3,3′,4′,5,7-pentahydroxy-flavanone; diquertin;taxifoliol; and distylin. (+)-Taxifolin is well know and appreciated inthe art of pharmaceutical arts and is readily available commercially.

The preferred p-glycoprotein inhibitor for use in the present inventionare water soluble vitamin E, such as vitamin E TPGS, and thepolyethylene glycols. Of the polyethylene glycols, the most preferredp-glycoprotein inhibitors are PEG 400, PEG 1000, PEG 1450, PEG 4600 andPEG 8000.

Administration of a p-glycoprotein inhibitor may be by any route bywhich the p-glycoprotein inhibitor will be bioavailable in effectiveamounts including oral and parenteral routes. Although oraladministration is preferred, the p-glycoprotein inhibitors may also beadministered intravenously, topically, subcutaneously, intranasally,rectally, intramuscularly, or by other parenteral routes. Whenadministered orally, the p-glycoprotein inhibitor may be administered inany convenient dosage form including, for example, capsule, tablet,liquid, suspension, and the like.

Generally, an effective p-glycoprotein inhibiting amount of ap-glycoprotein inhibitor is that amount which is effective in providinginhibition of the activity of the p-glycoprotein mediated activetransport system present in the gut. An effective p-glycoproteininhibiting amount can vary between about 5 mg to about 1000 mg ofp-glycoprotein inhibitor as a daily dose depending upon the particularp-glycoprotein inhibitor selected, the species of patient to be treated,the dosage regimen, and other factors which are all well within theabilities of one of ordinary skill in the medical arts to evaluate andassess. A preferred amount however will typically be from about 50 mg toabout 500 mg, and a more preferred amount will typically be from about100 mg to about 500 mg. The above amounts of a p-glycoprotein inhibitorcan be administered from once to multiple times per day. Typically fororal dosing, doses will be administered on a regimen requiring one, twoor three doses per day.

Where water soluble vitamin E or a polyethylene glycol is selected asthe p-glycoprotein inhibitor, a preferred amount will typically be fromabout 5 mg to about 1000 mg, a more preferred amount will typically befrom about 50 mg to about 500 mg, and a further preferred amount willtypically be from about 100 mg to about 500 mg. The most preferredamount of water soluble vitamin E or a polyethylene glycol will be fromabout 200 mg to about 500 mg. The above amounts of water soluble vitaminE or polyethylene glycol can be administered from once to multiple timesper day. Typically, doses will be administered on a regimen requiringone, two or three doses per day with one and two being preferred.

As used herein, the term “co-administration” refers to administration toa patient of both a compound that has vasodilating and/or plateletaggregation inhibiting properties, including the compounds described inU.S. Pat. Nos. 4,306,075 and 5,153,222 which include treprostinil andstructures I and II described herein, and a p-glycoprotein inhibitor sothat the pharmacologic effect of the p-glycoprotein inhibitor ininhibiting p-glycoprotein mediated transport in the gut is manifest atthe time at which the compound is being absorbed from the gut. Ofcourse, the compound and the p-glycoprotein inhibitor may beadministered at different times or concurrently. For example, thep-glycoprotein inhibitor may be administered to the patient at a timeprior to administration of the therapeutic compound so as to pre-treatthe patient in preparation for dosing with the vasodilating compound.Furthermore, it may be convenient for a patient to be pre-treated withthe p-glycoprotein inhibitor so as to achieve steady state levels ofp-glycoprotein inhibitor prior to administration of the first dose ofthe therapeutic compound. It is also contemplated that the vasodilatingand/or platelet aggregation inhibiting compounds and the p-glycoproteininhibitor may be administered essentially concurrently either inseparate dosage forms or in the same oral dosage form.

The present invention further provides that the vasodilating and/orplatelet aggregation inhibiting compound and the p-glycoproteininhibitor may be administered in separate dosage forms or in the samecombination oral dosage form. Co-administration of the compound and thep-glycoprotein inhibitor may conveniently be accomplished by oraladministration of a combination dosage form containing both the compoundand the p-glycoprotein inhibitor.

Thus, an additional embodiment of the present invention is a combinationpharmaceutical composition for oral administration comprising aneffective vasodilating and/or platelet aggregation inhibiting amount ofa compound described herein and an effective p-glycoprotein inhibitingamount of a p-glycoprotein inhibitor. This combination oral dosage formmay provide for immediate release of both the vasodilating and/orplatelet aggregation inhibiting compound and the p-glycoproteininhibitor or may provide for sustained release of one or both of thevasodilating and/or platelet aggregation inhibiting compound and thep-glycoprotein inhibitor. One skilled in the art would readily be ableto determine the appropriate properties of the combination dosage formso as to achieve the desired effect of co-administration of thevasodilating and/or platelet aggregation inhibiting compound and thep-glycoprotein inhibitor.

Accordingly, the present invention provides for an enhancement of thebioavailability of treprostinil, a drug of structure I or II, andpharmaceutically acceptable salts thereof by co-administration of ap-glycoprotein inhibitor. By co-administration of these compounds and ap-glycoprotein inhibitor, the total amount of the compound can beincreased over that which would otherwise circulate in the blood in theabsence of the p-glycoprotein inhibitor. Thus, co-administration inaccordance with the present invention can cause an increase in the AUCof the present compounds over that seen with administration of thecompounds alone.

Typically, bioavailability is assessed by measuring the drugconcentration in the blood at various points of time afteradministration of the drug and then integrating the values obtained overtime to yield the total amount of drug circulating in the blood. Thismeasurement, called the Area Under the Curve (AUC), is a directmeasurement of the bioavailability of the drug.

Without limiting the scope of the invention, it is believed that in someembodiments derivatizing treprostinil at the R² and R³ hydroxyl groupscan help overcome the barriers to oral treprostinil delivery by blockingthese sites, and thus the metabolism rate may be reduced to permit thecompound to bypass some of the first pass effect. Also, with an exposedamino acid, the prodrug may be actively absorbed from the dipeptidetransporter system that exists in the gastrointestinal tract.Accordingly, the present invention provides compounds, such as thosefound in structures I and II, that reduce the first pass effect oftreprostinil and/or reduce the efflux mechanism of the gastrointestinaltract.

In some embodiments of the method of treating hypertension in a subject,the subject is a mammal, and in some embodiments is a human.

Pharmaceutical formulations may include any of the compounds of any ofthe embodiments described above, either alone or in combination, incombination with a pharmaceutically acceptable carrier such as thosedescribed herein.

The instant invention also provides for compositions which may beprepared by mixing one or more compounds of the instant invention, orpharmaceutically acceptable salts thereof, with pharmaceuticallyacceptable carriers, excipients, binders, diluents or the like, to treator ameliorate a variety of disorders related vasoconstriction and/orplatelet aggregation. A therapeutically effective dose further refers tothat amount of one or more compounds of the instant invention sufficientto result in amelioration of symptoms of the disorder. Thepharmaceutical compositions of the instant invention can be manufacturedby methods well known in the art such as conventional granulating,mixing, dissolving, encapsulating, lyophilizing, emulsifying orlevigating processes, among others. The compositions can be in the formof, for example, granules, powders, tablets, capsules, syrup,suppositories, injections, emulsions, elixirs, suspensions or solutions.The instant compositions can be formulated for various routes ofadministration, for example, by oral administration, by transmucosaladministration, by rectal administration, transdermal or subcutaneousadministration as well as intrathecal, intravenous, intramuscular,intraperitoneal, intranasal, intraocular or intraventricular injection.The compound or compounds of the instant invention can also beadministered by any of the above routes, for example in a local ratherthan a systemic fashion, such as injection as a sustained releaseformulation. The following dosage forms are given by way of example andshould not be construed as limiting the instant invention.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant invention, or pharmaceuticallyacceptable salts thereof, with at least one additive or excipient suchas a starch or other additive. Suitable additives or excipients aresucrose, lactose, cellulose sugar, mannitol, maltitol, dextran,sorbitol, starch, agar, alginates, chitins, chitosans, pectins,tragacanth gum, gum arabic, gelatins, collagens, casein, albumin,synthetic or semi-synthetic polymers or glycerides, methyl cellulose,hydroxypropylmethyl-cellulose, and/or polyvinylpyrrolidone. Optionally,oral dosage forms can contain other ingredients to aid inadministration, such as an inactive diluent, or lubricants such asmagnesium stearate, or preservatives such as paraben or sorbic acid, oranti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Additionally, dyestuffs orpigments may be added for identification. Tablets may be further treatedwith suitable coating materials known in the art.

Additionally, tests have shown that the present compounds, includingtreprostinil, and in particular the compounds of structure I and II haveincreased bioavailability when delivered to the duodenum. Accordingly,one embodiment of the present invention involves preferential deliveryof the desired compound to the duodenum as well as pharmaceuticalformulations that achieve duodenal delivery. Duodenal administration canbe achieved by any means known in the art. In one of these embodiments,the present compounds can be formulated in an enteric-coated dosageform. Generally, enteric-coated dosage forms are usually coated with apolymer that is not soluble at low pH, but dissolves quickly whenexposed to pH conditions of 3 or above. This delivery form takesadvantage of the difference in pH between the stomach, which is about 1to 2, and the duodenum, where the pH tends to be greater than 4.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions,slurries and solutions, which may contain an inactive diluent, such aswater. Pharmaceutical formulations may be prepared as liquid suspensionsor solutions using a sterile liquid, such as, but not limited to, anoil, water, an alcohol, and combinations of these. Pharmaceuticallysuitable surfactants, suspending agents, emulsifying agents, may beadded for oral or parenteral administration.

As noted above, suspensions may include oils. Such oil include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Preferably, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation may be a powder suitablefor reconstitution with an appropriate solution as described above.Examples of these include, but are not limited to, freeze dried, rotarydried or spray dried powders, amorphous powders, granules, precipitates,or particulates. For injection, the formulations may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these. The compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampoules or inmulti-dose containers.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carries are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

The formulations of the invention may be designed for to beshort-acting, fast-releasing, long-acting, and sustained-releasing asdescribed below. Thus, the pharmaceutical formulations may also beformulated for controlled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations may be compressedinto pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

A therapeutically effective dose may vary depending upon the route ofadministration and dosage form. The preferred compound or compounds ofthe instant invention is a formulation that exhibits a high therapeuticindex. The therapeutic index is the dose ratio between toxic andtherapeutic effects which can be expressed as the ratio between LD₅₀ andED₅₀. The LD₅₀ is the dose lethal to 50% of the population and the ED₅₀is the dose therapeutically effective in 50% of the population. The LD₅₀and ED₅₀ are determined by standard pharmaceutical procedures in animalcell cultures or experimental animals.

A method of preparing pharmaceutical formulations includes mixing any ofthe above-described compounds with a pharmaceutically acceptable carrierand water or an aqueous solution.

Pharmaceutical formulations and medicaments according to the inventioninclude any of the compounds of any of the embodiments of compound ofstructure I, II or pharmaceutically acceptable salts thereof describedabove in combination with a pharmaceutically acceptable carrier. Thus,the compounds of the invention may be used to prepare medicaments andpharmaceutical formulations. In some such embodiments, the medicamentsand pharmaceutical formulations comprise any of the compounds of any ofthe embodiments of the compounds of structure I or pharmaceuticallyacceptable salts thereof. The invention also provides for the use of anyof the compounds of any of the embodiments of the compounds of structureI, II or pharmaceutically acceptable salts thereof for prostacyclin-likeeffects. The invention also provides for the use of any of the compoundsof any of the embodiments of the compounds of structure I, II orpharmaceutically acceptable salts thereof or for the treatment ofpulmonary hypertension.

The invention also pertains to kits comprising one or more of thecompounds of structure I or II along with instructions for use of thecompounds. In another embodiment, kits having compounds withprostacyclin-like effects described herein in combination with one ormore p-glycoprotein inhibitors is provided along with instructions forusing the kit.

By way of illustration, a kit of the invention may include one or moretablets, capsules, caplets, gelcaps or liquid formulations containingthe bioenhancer of the present invention, and one or more tablets,capsules, caplets, gelcaps or liquid formulations containing aprostacyclin-like effect compound described herein in dosage amountswithin the ranges described above. Such kits may be used in hospitals,clinics, physician's offices or in patients' homes to facilitate theco-administration of the enhancing and target agents. The kits shouldalso include as an insert printed dosing information for theco-administration of the enhancing and target agents.

The following abbreviations and definitions are used throughout thisapplication:

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium.

As used herein, the term “p-glycoprotein inhibitor” refers to organiccompounds which inhibit the activity of the p-glycoprotein mediatedactive transport system present in the gut. This transport systemactively transports drugs which have been absorbed from the intestinallumen and into the gut epithelium back out into the lumen Inhibition ofthis p-glycoprotein mediated active transport system will cause lessdrug to be transported back into the lumen and will thus increase thenet drug transport across the gut epithelium and will increase theamount of drug ultimately available in the blood.

The phrases “oral bioavailability” and “bioavailability upon oraladministration” as used herein refer to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered orally to apatient.

The phrase “unsubstituted alkyl” refers to alkyl groups that do notcontain heteroatoms. Thus the phrase includes straight chain alkylgroups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase alsoincludes branched chain isomers of straight chain alkyl groups,including but not limited to, the following which are provided by way ofexample: —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃,—C(CH₂CH₃)₃, —CH₂CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂,—CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The phrase also includescyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted withstraight and branched chain alkyl groups as defined above. The phrasealso includes polycyclic alkyl groups such as, but not limited to,adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings substitutedwith straight and branched chain alkyl groups as defined above. Thus,the phrase unsubstituted alkyl groups includes primary alkyl groups,secondary alkyl groups, and tertiary alkyl groups. Unsubstituted alkylgroups may be bonded to one or more carbon atom(s), oxygen atom(s),nitrogen atom(s), and/or sulfur atom(s) in the parent compound.Preferred unsubstituted alkyl groups include straight and branched chainalkyl groups and cyclic alkyl groups having 1 to 20 carbon atoms. Morepreferred such unsubstituted alkyl groups have from 1 to 10 carbon atomswhile even more preferred such groups have from 1 to 5 carbon atoms.Most preferred unsubstituted alkyl groups include straight and branchedchain alkyl groups having from 1 to 3 carbon atoms and include methyl,ethyl, propyl, and —CH(CH₃)₂.

The phrase “substituted alkyl” refers to an unsubstituted alkyl group asdefined above in which one or more bonds to a carbon(s) or hydrogen(s)are replaced by a bond to non-hydrogen and non-carbon atoms such as, butnot limited to, a halogen atom in halides such as F, Cl, Br, and I; andoxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxygroups, and ester groups; a sulfur atom in groups such as thiol groups,alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, andsulfoxide groups; a nitrogen atom in groups such as amines, amides,alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, and enamines; a silicon atom in groups such as intrialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups,and triarylsilyl groups; and other heteroatoms in various other groups.Substituted alkyl groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom is replaced by a bond to a heteroatomsuch as oxygen in carbonyl, carboxyl, and ester groups; nitrogen ingroups such as imines, oximes, hydrazones, and nitriles. Preferredsubstituted alkyl groups include, among others, alkyl groups in whichone or more bonds to a carbon or hydrogen atom is/are replaced by one ormore bonds to fluorine atoms. One example of a substituted alkyl groupis the trifluoromethyl group and other alkyl groups that contain thetrifluoromethyl group. Other alkyl groups include those in which one ormore bonds to a carbon or hydrogen atom is replaced by a bond to anoxygen atom such that the substituted alkyl group contains a hydroxyl,alkoxy, aryloxy group, or heterocyclyloxy group. Still other alkylgroups include alkyl groups that have an amine, alkylamine,dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine,heterocyclylamine, (alkyl)(heterocyclyl)amine,(aryl)(heterocyclyl)amine, or diheterocyclylamine group.

The phrase “unsubstituted arylalkyl” refers to unsubstituted alkylgroups as defined above in which a hydrogen or carbon bond of theunsubstituted alkyl group is replaced with a bond to an aryl group asdefined above. For example, methyl (—CH₃) is an unsubstituted alkylgroup. If a hydrogen atom of the methyl group is replaced by a bond to aphenyl group, such as if the carbon of the methyl were bonded to acarbon of benzene, then the compound is an unsubstituted arylalkyl group(i.e., a benzyl group). Thus the phrase includes, but is not limited to,groups such as benzyl, diphenylmethyl, and 1-phenylethyl(—CH(C₆H₅)(CH₃)) among others.

The phrase “substituted arylalkyl” has the same meaning with respect tounsubstituted arylalkyl groups that substituted aryl groups had withrespect to unsubstituted aryl groups. However, a substituted arylalkylgroup also includes groups in which a carbon or hydrogen bond of thealkyl part of the group is replaced by a bond to a non-carbon or anon-hydrogen atom. Examples of substituted arylalkyl groups include, butare not limited to, —CH₂C(═O)(C₆H₅), and —CH₂(2-methylphenyl) amongothers.

A “pharmaceutically acceptable salt” includes a salt with an inorganicbase, organic base, inorganic acid, organic acid, or basic or acidicamino acid. As salts of inorganic bases, the invention includes, forexample, alkali metals such as sodium or potassium; alkaline earthmetals such as calcium and magnesium or aluminum; and ammonia. As saltsof organic bases, the invention includes, for example, trimethylamine,triethylamine, pyridine, picoline, ethanolamine, diethanolamine, andtriethanolamine. As salts of inorganic acids, the instant inventionincludes, for example, hydrochloric acid, hydroboric acid, nitric acid,sulfuric acid, and phosphoric acid. As salts of organic acids, theinstant invention includes, for example, formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, lactic acid, tartaricacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.As salts of basic amino acids, the instant invention includes, forexample, arginine, lysine and ornithine. Acidic amino acids include, forexample, aspartic acid and glutamic acid.

“Treating” within the context of the instant invention, means analleviation of symptoms associated with a biological condition,disorder, or disease, or halt of further progression or worsening ofthose symptoms, or prevention or prophylaxis of the disease or disorder.For example, within the context of treating patients having pulmonaryhypertension, successful treatment may include a reduction directvasodilation of pulmonary and/or systemic arterial vascular beds andinhibition of platelet aggregation. The result of this vasodilation willgenerally reduce right and left ventricular afterload and increasedcardiac output and stroke volume. Dose-related negative inotropic andlusitropic effects can also result. The outward manifestation of thesephysical effects can include a decrease in the symptoms of hypertension,such as shortness of breath, and an increase in exercise capacity.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1

In this Example, the bioavailability of treprostinil in rats afterdosing orally, intraduodenally, intracolonically and via the portal veinwas compared to determine possible barriers to bioavailability. Inaddition to bioavailability, a number of pharmacokinetic parameters weredetermined.

Animal Dosing

The bioavailability of treprostinil was evaluated in Sprague-Dawley,male rats. Fifteen surgically modified rats were purchased from HilltopLab Animals (Scottdale, Pa.). The animals were shipped from Hilltop toAbsorption Systems' West Chester University facility (West Chester,Pa.), where they were housed for at least twenty-four hours prior tobeing used in the study. The animals were fasted for approximately 16hours prior to dosing. The fifteen rats used in this study were dividedinto five groups (I, II, III, IV and V).

The weight of the animals and the dosing regimen are presented in Table1.

TABLE 1 Dose Weight Route of Study Volume Dose Group Rat # (g)Administration Day (mL/kg) (mg/kg) I 118 327 Intravenous 0 2 1 119 329Intravenous 0 2 1 120 320 Intravenous 0 2 1 II 121 337 Intraportal Vein0 2 1 122 319 Intraportal Vein 0 2 1 123 330 Intraportal Vein 0 2 1 III124 329 Intraduodenal 0 2 1 125 331 Intraduodenal 0 2 1 126 324Intraduodenal 0 2 1 IV 127 339 Intracolonic 0 2 1 128 333 Intracolonic 02 1 129 320 Intracolonic 0 2 1 V 130 293 Oral 0 2 1 131 323 Oral 0 2 1132 332 Oral 0 2 1

Samples were withdrawn at the following time points.

-   -   IV and IPV: 0 (pre-dose) 2, 5, 15, 30, 60, 120, 240, 360, 480        minutes    -   ID, IC and Oral: 0 (pre-dose), 5, 15, 30, 60, 120, 240, 360, 480        minutes

Approximately 0.50 to 0.75 mL of whole blood was collected from thejugular vein of a cannulated rat. The blood was transferred toheparinized tubes and placed on ice until centrifuged. Followingcentrifugation the plasma was placed on ice until frozen at −70 C priorto shipment to Absorption Systems

Analysis of Plasma Samples

Samples were analyzed using the following methodology:

Dosing Solution Preparation

The dosing solution was prepared by combining 15.2 mg of treprostinildiethanolamine (12.0 mg of the free acid form) with 24 mL of 5%dextrose. The solution was then sonicated until dissolved for a finalconcentration of 0.5 mg/mL. The final pH of the dosing solution was 4.6.At the time of dosing, the dosing solution was clear and homogenous.

Standards and Sample Preparation

To determine the concentration of treprostinil in rat plasma samples,standards were prepared with rat plasma collected in heparin obtainedfrom Lampire Biological Laboratories (Lot #021335263) to contain 1000,300, 100, 30, 10, 3, 1 and 0.3 ng/mL of treprostinil. Plasma standardswere treated identically to the plasma samples.

Plasma samples were prepared by solid phase extraction. After anextraction plate was equilibrated, 150 μL of a plasma sample was placedinto the well and vacuumed through. The extraction bed was then washedwith 600 μL of acetonitrile: deionized water (25:75) with 0.2% formicacid. The compound was eluted with 600 μL of 90% acetonitrile and 10%ammonium acetate. The eluates were collected and evaporated to dryness.The residue was reconstituted with 150 μL of acetonitrile: deionizedwater (50:50) with 0.5 μg/mL of tolbutamide (used as an internalstandard).

HPLC Conditions

Column: Keystone Hypersil BDS C18 30×2 mm i.d., 3 μm.

Mobile Phase Buffer: 25 mM NH₄OH to pH 3.5 w/85% formic acid.

Reservoir A: 10% buffer and 90% water.

Reservoir B: 10% buffer and 90% acetonitrile.

Mobile Phase Composition:

Gradient Program:

Time Duration Grad. Curve % A % B −0.1 0.10 0 80 20 0 3.00 1.0 10 903.00 1.00 1.0 0 100 4.00 2.00 0 80 20

-   -   Flow Rate: 300 μL/min.    -   Inj. Vol.: 10 μL    -   Run Time: 6.0 min.    -   Retention Time: 2.6 min.    -   Mass Spectrometer    -   Instrument: PE SCIEX API 2000    -   Interface: Electrospray (“Turbo Ion Spray”)    -   Mode: Multiple Reaction Monitoring (MRM)

Precursor Ion Product Ion Treprostinil 389.2 331.2 IS 269.0 170.0

Nebulizing Gas: 25 Drying Gas: 60, 350° C. Curtain Gas: 25 Ion Spray:−5000 V Orifice: −80 V Ring: −350 V Q0: 10 V IQ1: 11 V ST: 15 V R01: 11V IQ2: 35 V R02: 40 V IQ3: 55 V R03: 45 V CAD Gas: 4

Method Validation

Table 2 lists the average recoveries (n=6) and coefficient of variation(c.v.) for rat plasma spiked with treprostinil. All samples werecompared to a standard curve prepared in 50:50 dH₂O:acetonitrile with0.5 μg/mL of tolbutamide to determine the percent of treprostinilrecovered from the plasma.

TABLE 2 Accuracy and Precision of Method Spiked Concentration PercentRecovered Coefficient of Variation 1000 ng/mL 85.6 5.2 100 ng/mL 89.611.6 10 ng/mL 98.8 7.0

Pharmacokinetic Analysis

Pharmacokinetic analysis was performed on the average plasmaconcentration for each time point.

The data were subjected to non-compartmental analysis using thepharmacokinetic program WinNonlin v. 3.1 (2).

Results Clinical Observations

Prior to beginning the experiments it was noted thatsupra-pharmacological doses of treprostinil would be needed to achieveplasma concentrations that could be analyzed with adequate sensitivity.Using the dose of 1 mg/kg some adverse effects were noted in animalsdosed intravenously and via the intraportal vein.

All rats dosed intravenously displayed signs of extreme lethargy fiveminutes after dosing but fully recovered to normal activity thirtyminutes post-dosing. In addition, fifteen minutes after dosing all threeanimals dosed via the portal vein exhibited signs of lethargy. One rat(#123) expired before the thirty-minute sample was drawn. The other ratsfully recovered. The remaining animals did not display any adversereactions after administration of the compound.

Sample Analysis

Average plasma concentrations for each route of administration are shownin Table 3.

TABLE 3 Average (n = 3) plasma concentrations (ng/mL) Pre- Time (min)dose 2 5 15 30 60 120 240 360 480 Intravenous 0 1047.96 364.28 130.9155.56 14.45 4.45 1.09 0.50 0.30 Intraportal Vein* 0 302.28 97.39 47.9821.94 11.06 3.87 2.51 4.95 5.14 Intraduodenal 0 — 61.76 31.67 18.5713.55 5.91 1.11 0.89 0.90 Intracolonic 0 — 7.46 3.43 3.52 1.48 0.64 0.360.06^(λ) 0.20^(λ) Oral 0 — 4.52 2.90 3.67 2.06 4.52 1.82 0.90 0.96 *n =2, ^(λ)concentration falls below the limit of quantitation (LOQ) of theanalytical method

The plasma concentration versus time curves for intravenous,intraportal, intraduodenal, intracolonic and oral dosing are shown inFIGS. 1 and 2. FIG. 3 shows the average plasma concentration versus timecurves for all five routes of administration. In the experiments shownin these figures, the diethanolamine salt was used. Table 4 shows thepharmacokinetic parameters determined for treprostinil. The individualbioavailabilities of each rat are found in Table 5.

TABLE 4 Average Bioavailability and Pharmacokinetic Parameters ofTreprostinil in Rats Average Average Volume of CLs Route ofAUC_(480 min) C_(max) T_(max) T_(1/2) Bioavailability Distribution* (mL· min⁻¹ · Administration (min · ng/mL) (ng/mL) (min) (min) (%) ± SD (L ·kg⁻¹) kg⁻¹)* Intravenous 11253.49 2120^(Ψ) 0 94 NA 1.98 88.54Intraportal Vein 4531.74  302 2 ND 40.3 ± 5.5 ND ND Intraduodenal2712.55  62 5 ND 24.1 ± 0.5 ND ND Intracolonic 364.63   8 5 ND  3.2 ±2.5 ND ND Oral 1036.23   5 5 ND  9.2 ± 1.4 ND ND *Normalized to theaverage weight of the rats ND: Not determined ^(Ψ)Extrapolated Value

TABLE 5 Individual Bioavailabilities of Treprostinil in Rats IndividualRoute of Individual AUC_(480 min) Bioavailability Administration Rat #(min · ng/mL) (%) Intravenous 118 10302.85 NA 119 9981.52 NA 12013510.65 NA Intraportal Vein 121 4970.67 44.2 122 4093.21 36.4 123 ND NDIntraduodenal 124 2725.68 24.2 125 2763.60 24.6 126 2646.05 23.5Intracolonic 127 72.63 0.7 128 395.08 3.5 129 625.20 5.6 Oral 130 998.708.9 131 907.60 8.1 132 1203.73 10.7 NA: Not applicable ND: Notdetermined

Conclusions

Treprostinil has a terminal plasma half-life of 94 minutes. Thedistribution phase of treprostinil has a half-life of 10.3 minutes andover 90% of the distribution and elimination of the compound occurs by60 minutes post-dosing. The volume of distribution (Vd=1.98 L/kg) isgreater than the total body water of the rat (0.67 L/kg) indicatingextensive partitioning into tissues. The systemic clearance oftreprostinil (88.54 mL/min/kg) is greater than the hepatic blood flowsignifying that extra-hepatic clearance mechanisms are involved in theelimination of the compound.

First pass hepatic elimination of treprostinil results in an averageintraportal vein bioavailability of 40.3%. Fast but incompleteabsorption is observed after intraduodenal, intracolonic and oral dosing(T_(max) 5 min). By comparing the intraportal vein (40.3%) andintraduodenal bioavailability (24.1%) it appears that approximately 60%of the compound is absorbed in the intestine. The average intraduodenalbioavailibility is almost three times greater than the oralbioavailibility suggesting that degradation of treprostinil in thestomach or gastric emptying may influence the extent of systemicabsorption.

Example 2

In this Example, Treprostinil concentrations were determined in maleSprague-Dawley rats following a single oral dose of the followingcompounds:

Experimental Dosing Solution Preparation

All dosing vehicles were prepared less than 2 hours prior to dosing.

1. Treprostinil Methyl Ester

A solution of treprostinil methyl ester was prepared by dissolving 2.21mg of treprostinil methyl ester with 0.85 mL of dimethylacetamide (DMA).This solution was then diluted with 7.65 mL of PEG 400: Polysorbate 80:Water, 40:1:49. The final concentration of the dosing vehicle was 0.26mg/mL of treprostinil methyl ester equivalent to 0.25 mg/mL ofTreprostinil. The dosing vehicle was a clear solution at the time ofdosing.

2. Treprostinil Benzyl Ester

A solution of treprostinil benzyl ester was prepared by dissolving 2.58mg of treprostinil benzyl ester with 0.84 mL of dimethylacetamide (DMA).This solution was then diluted with 7.54 mL of PEG 400: Polysorbate 80:Water, 40:1:49. The final concentration of the dosing vehicle was 0.268mg/mL of treprostinil benzyl ester equivalent to 0.25 mg/mL ofTreprostinil. The dosing vehicle was a clear solution at the time ofdosing.

3. Treprostinil Diglycine

A solution of treprostinil diglycine was prepared by dissolving 1.86 mgof compound with 0.58 mL of dimethylacetamide (DMA). This solution wasthen diluted with 5.18 mL of PEG 400: Polysorbate 80: Water, 40:1:49.The final concentration of the dosing vehicle was 0.323 mg/mL oftreprostinil diglycine equivalent to 0.25 mg/mL of Treprostinil. Thedosing vehicle was a clear solution at the time of dosing.

Animal Dosing

The plasma concentrations of Treprostinil following administration ofeach prodrug were evaluated in male Sprague-Dawley rats. Rats werepurchased from Hilltop Lab Animals (Scottdale, Pa.). The animals wereshipped from Hilltop to Absorption Systems' West Chester Universityfacility (West Chester, Pa.). They were housed for at least twenty-fourhours prior to being used in the study. The animals were fasted forapproximately 16 hours prior to dosing. The rats used in this study weredivided into three groups (I, II and III). Groups I-III were dosed onthe same day.

The weight of the animals and the dosing regimen are presented in Table6.

TABLE 6 Study Design Route of Compound Dose Volume Dose* Group Rat #Weight (kg) Administration Dosed (mL/kg) (mg/kg) I 638 306 OralTreprostinil 2 0.520 639 310 Oral methyl ester 640 319 Oral II 641 319Oral Treprostinil 2 0.616 642 309 Oral benzyl ester 643 320 Oral III 644318 Oral Treprostinil 2 0.646 645 313 Oral diglycine 646 322 Oral *Thisdose of prodrug = 0.500 mg/kg of the active, Treprostinil

Animals were dosed via oral gavage. Blood samples were taken from ajugular vein cannula at the following time points:

-   -   0 (pre-dose) 5, 15, 30, 60, 120, 240, 360 and 480 minutes

The blood samples were withdrawn and placed into tubes containing 30 μLof a solution of 500 units per mL of heparin in saline, and centrifugedat 13,000 rpm for 10 minutes. Approximately 200 μL of plasma was thenremoved and dispensed into appropriately labeled polypropylene tubescontaining 4 μL of acetic acid in order to stabilize any prodrugremaining in the samples. The plasma samples were frozen at −20° C. andwere transported on ice to Absorption Systems Exton Facility. There theywere stored in a −80° C. freezer pending analysis.

Analysis of Plasma Samples

Plasma samples were analyzed as described in Example 1. In brief,Treprostinil was extracted from the plasma via liquid-liquid extractionthen analyzed by LC/MS/MS. The analytical validation results werereported previously in Example 1. The lower limit of quantification(LLOQ) of the analytical method was 0.01 ng/mL. Samples were not assayedfor unchanged prodrug.

Acceptance Criteria for Analytical Runs

Two standard curves, with a minimum of five points per curve, and aminimum of two quality control samples (QCs) were dispersed throughouteach run. Each route of administration was bracketed by a standard curveused for back-calculation. The standards and QCs must be within ±15%(20% for the LLOQ) accuracy and precision for the run to be accepted. Atleast 75% of all standards and QCs must pass the acceptance criteria.

Pharmacokinetic Analysis

Pharmacokinetic analysis was performed on the plasma concentration ofTreprostinil for each individual rat at each time point and on theaverage plasma concentration for all three rats in the group for eachtime point. The data were subjected to non-compartmental analysis usingthe pharmacokinetic program WinNonLin v. 3.1 (2).

Results Study Observations

No adverse reactions were observed following oral administration oftreprostinil methyl ester, treprostinil benzyl ester or treprostinildiglycine.

Plasma Stability of Prodrugs in Acidified Rat Plasma

In order to terminate any conversion of prodrug to active after sampleswere withdrawn the plasma was acidified. Acetic acid (v/v) was added toeach plasma sample immediately after centrifugation of the red bloodcells to a concentration of 2%. In-vitro plasma stability of eachprodrug was performed to insure that the compound was stable inacidified plasma. To perform this assay 2% acetic acid was added toblank rat plasma obtained from Lampire Biological. The acidified ratplasma was equilibrated at 37° C. for three minutes prior to addition ofprodrug. The initial concentration of each prodrug was 1000 ng/mL. A 100μL aliquot of plasma (n=3 per time point) was taken at 0, 60 and 120minutes. Each aliquot was combined with 20 μL of HCl and vortexed.Liquid-liquid extraction was then performed and the concentration ofTreprostinil in each sample determined. The concentration ofTreprostinil at each time point in acidified rat plasma is given inTable 7. Small amounts of Treprostinil appear to be present in the neatcompound sample of treprostinil methyl ester and treprostinil diglycine.The concentration of Treprostinil remained constant throughout thecourse of the experiment, indicating that there was no conversion ofprodrug into active compound occurring in acidified plasma.

TABLE 7 Plasma Stability of Prodrugs in Acidified Dog PlasmaTreprostinil Concentration (ng/mL) ± SD (n = 3) TreprostinilTreprostinil Treprostinil Time (min) methyl ester benzyl ester diglycine0 56.8 ± 9.3 <0.01 54.9 ± 4.3 60 55.1 ± 5.0 <0.01 51.8 ± 5.9 120 53.8 ±1.3 <0.01 54.5 ± 0.8 Total % Treprostinil 5.7 <0.01 5.5

Average Treprostinil plasma concentrations following administration oftreprostinil methyl ester, treprostinil benzyl ester or treprostinildiglycine are shown in Table 8.

TABLE 8 Treprostinil Concentrations (Average ± SD (n = 3) PlasmaConcentrations (ng/mL) Oral Dosing Pre- 5 15 30 60 120 240 360 480Solution Dose (min) (min) (min) (min) (min) (min) (min) (min)Treprostinil 0 <0.01 0.2 ± 0.0 0.3 ± 0.1 0.5 ± 0.1 1.5 ± 0.8 0.2 ± 0.7<0.01 0.1 ± 0.1 methyl ester Treprostinil 0 3.1 ± 2.8 1.9 ± 0.8 2.5 ±1.5 3.2 ± 1.9 7.3 ± 4.9 1.6 ± 1.2 0.4 ± 0.40 0.6 ± 0.9 benzyl esterTreprostinil 0 <0.01 1.1 ± 1.9  6.6 ± 10.7  0.5 ± 0.3* 40. ± 5.8  9.0 ±13.5 2.1 ± 2.9  1.3 ± 0.8 diglycine *Due to insufficient amount ofsample collected this time point is the average of n = 2 rats.

FIGS. 4-7 contain graphical representations of the plasma concentrationversus time curves for Treprostinil in rat following administration ofeach prodrug. Table 9 lists each figure and the information displayed.

TABLE 9 List of Figures Figure Description 4 Oral Dose of Treprostinilmethyl ester 5 Oral Dose of Treprostinil benzyl ester 6 Oral Dose ofTreprostinil diglycine 7 Oral Dose of Treprostinil benzyl ester andTreprostinil diglycine Compared to Treprostinil Alone from Example 1

Pharmacokinetic Analysis

Bioavailability of the prodrug was determined relative to that of theactive compound based on Example 1 in which Treprostinil was dosed torats. The following formula was used to determine relativebioavailability (F):

RelativeF=(AUC_((Prodrug Dose))/Dose)/(AUC_((Treprostinil Dose))/Dose)*100

Bioavailability was also determined relative to an intravenous dose ofTreprostinil in rats determined in Example 1. Results are listed inTable 10.

TABLE 10 Average Relative Bioavailability and Pharmacokinetic Parametersof Treprostinil in Rats Test Average Relative Compound Dose AUC_(0-t)C_(max) T_(max) Bioavailability Bioavailability Administered (mg/kg)(min · ng/mL) (ng/mL) (min) (%) ± SD (n = 3) (%) ± SD (n = 3)Treprostinil 0.5 212 1.50 120 41.0 ± 16   3.8 ± 2 methyl esterTreprostinil 0.5 1171 7.20 120 226 ± 155 20.8 ± 14 benzyl esterTreprostinil 0.5 2242 9.04 240 433 ± 631 39.9 ± 58 diglycine

Conclusions

In this study the relative oral bioavailabilities of prodrugs ofTreprostinil were determined in rats. Treprostinil methyl ester resultedin Treprostinil area under the plasma concentration versus time curves(AUCs) less than that after dosing the active compound. Prodrugstreprostinil benzyl ester and treprostinil diglycine both hadTreprostinil average AUCs greater than that after dosing of the activecompound. Treprostinil diglycine had the highest relativebioavailability of 433% with over 4 times more Treprostinil reaching thesystemic circulation. The Cmax of 9 ng/mL of Treprostinil followingadministration of treprostinil diglycine occurred at 240 minutespost-dosing. The Cmax following dosing of Treprostinil is 5 ng/mL andoccurs only 5 minutes post-dosing. Treprostinil benzyl ester had arelative bioavailability of 226±155% with a Cmax of 7.2 ng/mL occurring120 minutes post-dosing. It should also be noted that the AUCs are notextrapolated to infinity.

REFERENCES

-   1. WinNonlin User's Guide, version 3.1, 1998-1999, Pharsight Co.,    Mountain View, Calif. 94040.

Example 3

This example illustrates a pharmacokinetic study of treprostinilfollowing administration of a single duodenal dose of treprostinil andvarious prodrugs of the present invention.

In this study, the area under the curve of Treprostinil in maleSprague-Dawley rats following a single intraduodenal dose oftreprostinil monophosphate (ring), treprostinil monovaline (ring),treprostinil monoalanine (ring) or treprostinil monoalanine (chain),prodrugs of treprostinil was compared. The compounds were as follows:

having the following substituents:

Compound R¹ R² R³ treprostinil H —PO₃H₃ H monophosphate (ring)treprostinil H —COCH(CH(CH₃)₂)NH₂ H monovaline (ring) treprostinil H—COCH(CH₃)NH₂ H monoalanine (ring) treprostinil H H —COCH(CH₃)NH₂monoalanine (chain)

Experimental Dosing Solution Preparation

All dosing vehicles were prepared less than 2 hours prior to dosing.

1. Treprostinil Monophosphate (Ring)

A dosing solution of treprostinil monophosphate (ring) was prepared bydissolving 1.01 mg of treprostinil monophosphate (ring) in 0.167 mL ofdimethylacetamide (DMA) until dissolved. This solution was furtherdiluted with 1.50 mL of PEG 400: Polysorbate 80: Water, 40:1:49. Thefinal concentration of the dosing vehicle was 0.603 mg/mL of prodrugequivalent to 0.5 mg/mL of Treprostinil. The dosing vehicle was a clearsolution at the time of dosing.

2. Treprostinil Monovaline (Ring)

A 50 mg/mL solution of treprostinil monovaline (ring) was prepared indimethylacetamide (DMA). A 25 μL aliquot of the 50 mg/mL stock solutionwas then diluted with 175 μL of DMA and 1.8 mL of PEG 400: Polysorbate80: Water, 40:1:49. The final concentration of the dosing vehicle was0.625 mg/mL of prodrug equivalent to 0.5 mg/mL of Treprostinil. Thedosing vehicle was a clear solution at the time of dosing.

3. Treprostinil Monoalanine (Ring)

A solution of treprostinil monoalanine (ring) was prepared by dissolving1.05 mg of treprostinil monoalanine (ring) in 0.178 mL ofdimethylacetamide (DMA) until dissolved. This solution was furtherdiluted with 1.60 mL of PEG 400: Polysorbate 80: Water, 40:1:49. Thefinal concentration of the dosing vehicle was 0.590 mg/mL oftreprostinil monoalanine (ring) equivalent to 0.5 mg/mL of Treprostinil.The dosing vehicle was a clear solution at the time of dosing.

4. Treprostinil Monoalanine (Chain)

A solution of treprostinil monoalanine (chain) was prepared bydissolving 0.83 mg of treprostinil monoalanine (chain) in 0.14 mL ofdimethylacetamide (DMA) until dissolved. This solution was furtherdiluted with 1.26 mL of PEG 400: Polysorbate 80: Water, 40:1:49. Thefinal concentration of the dosing vehicle was 0.591 mg/mL oftreprostinil monoalanine (chain) equivalent to 0.5 mg/mL ofTreprostinil. The dosing vehicle was a clear solution at the time ofdosing.

Animal Dosing

The plasma concentrations of Treprostinil following oral administrationof each prodrug were evaluated in male Sprague-Dawley rats. Twelve ratswere purchased from Hilltop Lab Animals (Scottdale, Pa.). The animalswere shipped from Hilltop to Absorption Systems' West Chester Universityfacility (West Chester, Pa.). They were housed for at least twenty-fourhours prior to being used in the study. The animals were fasted forapproximately 16 hours prior to dosing. The twelve rats used in thisstudy were divided into four groups. All groups were dosed on day 1 ofthe study. The weight of the animals and the dosing regimen arepresented in Table 11.

TABLE 11 Dose Rat Weight Volume Dose* # (g) Compound (mL/kg) (mg/kg) 130327 treprostinil monophosphate (ring) 1 0.603 131 321 treprostinilmonophosphate (ring) 1 0.603 132 310 treprostinil monophosphate (ring) 10.603 133 328 treprostinil monovaline (ring) 1 0.625 134 326treprostinil monovaline (ring) 1 0.625 135 346 treprostinil monovaline(ring) 1 0.625 136 321 treprostinil monoalanine (chain) 1 0.591 137 319treprostinil monoalanine (chain) 1 0.591 138 330 treprostinilmonoalanine (chain) 1 0.591 139 316 treprostinil monoalanine (ring) 10.590 140 330 treprostinil monoalanine (ring) 1 0.590 141 339treprostinil monoalanine (ring) 1 0.590 *This dose of prodrug = 0.500mg/kg of treprostinil

Animals were dosed via an indwelling duodenal cannula. Blood sampleswere taken from a jugular vein cannula at the following time points: 0(pre-dose) 5, 15, 30, 60, 120, 240, 360 and 480 minutes.

The blood samples were withdrawn and placed into tubes containing 30 μLof a solution of 500 units per mL of heparin in saline, and centrifugedat 13,000 rpm for 10 minutes. Approximately 200 μL of plasma was thenremoved and dispensed into appropriately labeled polypropylene tubescontaining 4 μL of acetic acid in order to stabilize any prodrugremaining in the samples. The plasma samples were frozen at −20° C. andwere transported on ice to Absorption Systems Exton Facility. There theywere stored in a −80° C. freezer pending analysis.

Analysis of Plasma Samples

Plasma samples were analyzed using the methods described above. Inbrief, Treprostinil was extracted from the plasma via solid phaseextraction then analyzed by LC/MS/MS. The lower limit of quantification(LLOQ) of the analytical method was 0.03 ng/mL.

Acceptance Criteria for Analytical Runs

Four standard curves, with a minimum of five points per curve, and aminimum of two quality control samples (QCs) at 3 concentrations weredispersed throughout each run. Each prodrug set was bracketed by astandard curve used for back-calculation. The standards and QCs must bewithin ±15% (20% for the LLOQ) accuracy and precision for the run to beaccepted. At least 75% of all standards and QCs must pass the acceptancecriteria.

Pharmacokinetic Analysis

Pharmacokinetic analysis was performed on the plasma concentration ofTreprostinil for each individual rat at each time point and on theaverage plasma concentration for all three rats in the group for eachtime point.

The data were subjected to non-compartmental analysis using thepharmacokinetic program WinNonLin v. 3.1 (2).

Results Study Observations

No adverse reactions were observed following intraduodenaladministration of treprostinil monophosphate (ring), treprostinilmonovaline (ring), treprostinil monoalanine (ring) or treprostinilmonoalanine (chain).

Ex-Vivo Plasma Stability of Prodrugs in Acidified Rat Plasma

In order to terminate any conversion of prodrug to active after sampleswere withdrawn, the plasma was acidified. Acetic acid (v/v) was added toeach plasma sample immediately after separation of the red blood cellsto a concentration of 2%. In-vitro plasma stability of each prodrug wasperformed to insure that the compound was stable in acidified plasma. Toperform this assay 2% acetic acid was added to blank rat plasma obtainedfrom Lampire Biological. The acidified rat plasma was brought to roomtemperature for three minutes prior to addition of prodrug. The initialconcentration of each prodrug was 1000 ng/mL. A 100 μL aliquot of plasma(n=3 per time point) was taken at 0, 60 and 120 minutes. Samplepreparation of each plasma sample was performed as described above andthe concentration of Treprostinil monitored.

Treprostinil concentrations did not increase in any of the acidifiedplasma samples spiked with prodrug over the two-hour period of theexperiment.

Sample Analysis

Average Treprostinil plasma concentrations following administration oftreprostinil monophosphate (ring), treprostinil monovaline (ring),treprostinil monoalanine (ring) or treprostinil monoalanine (chain) areshown in Table 12.

TABLE 12 AVERAGE ± SD (N = 3) PLASMA TREPROSTINIL CONCENTRATIONS (NG/ML)Oral Dosing Pre- 5 15 30 60 120 240 360 480 Solution dose (min) (min)(min) (min) (min) (min) (min) (min) treprostinil 0 8.62 ± 3.0 6.57 ± 1.73.31 ± 1.2 4.31 ± 0.8 2.07 ± 0.4 0.91 ± 0.5 0.26 ± 0.08  0.3 ± 0.08monophosphate (ring) treprostinil 0 0.76 ± 0.2 0.91 ± 0.7 1.52 ± 0.61.53 ± 0.6 1.65 ± 0.7 0.66 ± 0.1 0.15 ± 0.03  0.05 ± 0.02 monovaline(ring) treprostinil 0 2.42 ± 0.6 2.52 ± 0.4 2.91 ± 0.6 3.25 ± 1.5 1.69 ±0.4 0.55 ± 0.2 0.20 ± 0.1  0.22 ± 0.2 monoalanine (ring) treprostinil 09.53 ± 2.6 3.92 ± 0.6 3.83 ± 0.7 2.74 ± 0.9 0.86 ± 0.4 0.29 ± 0.2 0.08 ±0.04 0.19 ± 0.3 monoalanine (chain)

FIGS. 8-12 contain graphical representations of the plasma concentrationversus time curves for Treprostinil in rat following administration ofeach prodrug. Table 13 lists each figure and the information displayed.

TABLE 13 Figure Description 8 Intraduodenal dose of treprostinilmonophosphate (ring) 9 Intraduodenal dose of treprostinil monovaline(ring) 10 Intraduodenal dose of treprostinil monoalanine (ring) 11Intraduodenal dose of treprostinil monoalanine (chain) 12 Intraduodenaldose of each prodrug compared to treprostinil alone from Example 1

Pharmacokinetic Analysis

Bioavailability of the prodrug was determined relative to that of theactive compound based on a previous study in which Treprostinil wasdosed to rats. The following formula was used to determine relativebioavailability (F):

RelativeF=(AUC_((Prodrug Dose))/Dose)/(AUC_((Treprostinil Dose))/Dose)*100

Absolute bioavailability was also estimated using data from anintravenous dose of Treprostinil in rats determined in Example 1.Results are listed in Table 14.

TABLE 14 List of Figures Figure Description 8 Intraduodenal Dose oftreprostinil monophosphate (ring) 9 Intraduodenal Dose of treprostinilmonovaline (ring) 10 Intraduodenal Dose of treprostinil monoalanine(ring) 11 Intraduodenal Dose of treprostinil monoalanine (chain) 12Intraduodenal Dose of Each Prodrug Compared to Treprostinil Alone fromExample 1

Conclusions

The relative intraduodenal bioavailabilities of four prodrugs ofTreprostinil were determined in rats. All the compounds had relativeintraduodenal bioavailabilities less than that of the active compound.treprostinil monophosphate (ring) and treprostinil monoalanine (ring)had the highest relative intraduodenal bioavailability at 56% and 38%respectively. The T_(max) for treprostinil monophosphate (ring) andtreprostinil monoalanine (chain) occurred minutes post-dosing.treprostinil monovaline (ring) and treprostinil monoalanine (ring) hadlonger absorption times with T_(max) values of 120 and 60 minutesrespectively. Maximum Treprostinil concentrations were highest followingtreprostinil monophosphate (ring) and treprostinil monoalanine (chain)dosing. They reached approximately 9 ng/mL 5 minutes post-dosing. Thebioavailabilities are much greater when dosed intraduodenally than whendosed orally as measured by treprostinil plasma levels.

REFERENCES

-   1. WinNonlin User's Guide, version 3.1, 1998-1999, Pharsight Co.,    Mountain View, Calif. 94040.

Example 4

In this Example, Treprostinil concentrations will be determined in maleSprague-Dawley rats following a single oral or intraduodenal dose of thefollowing compounds of structure II:

having the following substituents:

Cpd. R¹ R² R³ A —CH₂CONH₂ H H B —CH₂CON(CH₂)₂OH H H C —CH₂CON(CH₃)₂ H HD —CH₂CONHOH H H E —CH₂C₆H₄NO₂ (p)* H H F —CH₂C₆H₄OCH₃ (p)* H H G—CH₂C₆H₄Cl (o)* H H H —CH₂C₆H₄(NO₂)₂ (o, p)* H H I —CH₂C₆H₄F (p)* H H JH —PO₃H₃ H K H H —PO₃H₃ L H —COCH₂NH₂ H M H H —COCH₂NH₂ N H—COCH(CH₃)NH₂ H O H H —COCH(CH₃)NH₂ P H —COCH(CH₃)NH₂ —COCH(CH₃)NH₂ *odenotes ortho substitution, m denotes meta substitution and p denotespara substitution.

Examples of these compounds include:

Prodrug preparation and analysis will take place as described inExamples 1 and 2 above. Additionally, the oral bioavailability oftreprostinil, treprostinil sodium and the compounds shown in Example 2and this Example will be administered in close proximity to orsimultaneously with various different p-glycoprotein inhibitingcompounds at varying concentrations and tested to determine the effectof the p-glycoprotein inhibitors on the oral bioavailability of thecompounds. The p-glycoprotein inhibitors will be administered bothintravenously and orally.

Example 5 Clinical Studies with Treprostinil Diethanolamine Introduction

Prior to proceeding directly into clinical studies with a sustainedrelease (SR) solid dosage form of UT-15C (treprostinil diethanolamine),a determination of the pharmacokinetics of an oral “immediate release”solution was performed. The first clinical study (01-101) evaluated theability of escalating doses of an oral solution of UT-15C to reachdetectable levels in plasma, potential dose-plasma concentrationrelationship, bioavailability and the overall safety of UT-15C.Volunteers were dosed with the solutions in a manner that simulated asustained release formulation releasing drug over approximately 8 hours.

The second clinical study (01-102) assessed the ability of two SR soliddosage form prototypes (i.e., 1. microparticulate beads in a capsuleand, 2. tablet) to reach detectable levels in plasma and the potentialinfluence of food on these plasma drug concentrations. The SR prototypeswere designed to release UT-15C over approximately an 8 hour timeperiod.

Details of the two clinical studies are described below.

Clinical Study 01-101 A Safety, Tolerability, and Pharmacokinetic Studyof Multiple Escalating Doses of UT-15C (Treprostinil Diethanolamine)Administered as an Oral Solution in Healthy Adult Volunteers (IncludingStudy of Bioavailability).

The oral solution of UT-15C was administered to 24 healthy volunteers toassess the safety and pharmacokinetic profile of UT-15C as well as itsbioavailability. To mimic a SR release profile, doses were administeredevery two hours for four doses at either 0.05 mg per dose (total=0.2mg), 0.125 mg per dose (total=0.5 mg), 0.25 mg per dose (total=1.0 mg),or 0.5 mg per dose (total=2.0 mg). Study endpoints included standardsafety assessments (adverse events, vital signs, laboratory parameters,physical examinations, and electrocardiograms) as well aspharmacokinetic parameters.

All subjects received all four scheduled doses and completed the studyin its entirety. Treprostinil plasma concentrations were detectable inall subjects following administration of an oral solution dose ofUT-15C. Both AUC_(inf) and C_(max) increased in a linear fashion withdose for each of the four dose aliquots. The highest concentrationobserved in this study was 5.51 ng/mL after the third 0.25 mg solutiondose aliquot of the 2.0 mg UT-15C total dose. Based on historicalintravenous treprostinil sodium data, the mean absolute bioavailabilityvalues for the 0.2 mg, 0.5 mg, 1.0 mg and 2.0 mg doses of UT-15C wereestimated to be 21%, 23%, 24% and 25%, respectively. The results of thisstudy are respectively shown in FIGS. 13A-13D.

UT-15C was well tolerated by the majority of subjects at all dosesgiven. There were no clinically significant, treatment emergent changesin hematology, clinical chemistry, urinalysis, vital signs, physicalexams, and ECGs. The most frequently reported adverse events wereflushing, headache, and dizziness. This safety profile with UT-15C(treprostinil diethanolamine) is consistent with the reported safetyprofile and product labeling of Remodulin (treprostinil sodium) andother prostacyclin analogs. Thus, changing the salt form of treprostinildid not result in any unexpected safety issues following the protocolspecified dosing regimen (i.e. single dose every 2 hours for four totaldoses on a single day).

Clinical Study 01-102 A Safety, Tolerability, and Pharmacokinetic StudyComparing a Single Dose of a Sustained Release Capsule and TabletFormulation of UT-15C (Treprostinil Diethanolamine) Administered toHealthy Adult Volunteers in the Fasted and Fed State

The 01-102 study was designed to evaluate and compare the safety andpharmacokinetic profiles of a (1) UT-15C SR tablet prototype and, (2)UT-15C SR capsule prototype (microparticulate beads in a capsule) inboth the fasted and fed state. Each of the SR dosage forms were designedto release UT-15C (1 mg) over an approximate 8-hour time period.Fourteen healthy adult volunteers were assigned to receive the SR tabletformulation while an additional fourteen volunteers were assigned toreceive the SR capsule formulation. Subjects were randomized to receivea single dose (1 mg) of their assigned SR prototype in both the fastedand fed state. A crossover design was employed with a seven day wash-outperiod separating the fed/fasted states. For the fed portion of thestudy, subjects received a high calorie, high fat meal. Study endpointsincluded standard safety assessments (adverse events, vital signs,laboratory parameters, physical examinations, and electrocardiograms) aswell as pharmacokinetic parameters.

All subjects administered UT-15C SR tablets and capsules had detectabletreprostinil plasma concentrations. Calculations of area under the curvefrom zero to twenty-four hours (AUC₀₋₂₄) indicate that total exposure toUT-15C SR occurred in the following order: Tablet Fed>CapsuleFasted>Tablet Fasted>Capsule Fed. FIG. 14 displays the pharmacokineticprofiles of the two formulations in the fasted and fed states.

UT-15C SR tablets and capsules were tolerated by the majority ofsubjects. All adverse events were mild to moderate in severity and weresimilar to those described in Study 01-101 and in Remodulin's productlabeling. Additionally, there were no treatment-emergent changes invital signs, laboratory parameters, physical examinations, orelectrocardiograms throughout the study.

These results demonstrate that detectable and potentially therapeuticdrug concentrations can be obtained from a solid dosage form of UT-15Cand that these concentrations can be maintained over an extended periodof time through sustained release formulation technology.

Polymorphs of Treprostinil Diethanolamine

Two crystalline forms of UT-15C were identified as well as an amorphousform. The first, which is metastable, is termed Form A. The second,which is thermodynamically more stable, is Form B. Each form wascharacterized and interconversion studies were conducted to demonstratewhich form was thermodynamically stable. Form A is made according to themethods in Table 15. Form B is made from Form A, in accordance with theprocedures of Table 16.

TABLE 15 XRPD Sample Solvent Conditions^(a) Habit/Description Result^(b)ID tetrahydro- FE opaque white solids; A 1440- furan morphology unknown,72-02 birefringent SE glassy transparent solids A (PO) 1440- 72-03 SC(60° translucent, colorless A 1440- C.) glassy sheets of material, 72-16birefringent Toluene slurry white solids; opaque A + B 1440- (RT), 6 dmasses of smaller 72-01 particles toluene: SC(60° white solids;spherical A 1480- IPA C.) clusters of fibers, 21-03 (11.4:1)birefringent Water FE opaque white solids; A 1440- morphology unknowm,72-07 birefringent SE opaque ring of solids, A + B 1440- birefringent72-08 freeze white, glassy transparent A + B 1480- dry solids 58-02water: FE opaque white solids; A + 1440- ethanol morphology unknown,11.5 pk 72-09 (1:1) birefringent FE clear and oily substance B 1480-with some opaque solids 79-02 SE glassy opaque ring of A 1440- solid72-10 ^(a)FE = fast evaporation; SE = slow evaporation; SC = slow cool^(b)IS = insufficient sample; PO = preferred orientation; LC = lowcrystallinity; pk = peak ^(c)XRPD = X-ray powder diffraction

TABLE 16 XRPD Sample Solvent Conditions Habit/Description Result IDethanol/ FE glassy appearing solids —^(b) 1519- water of unknownmorphology; 68-01 (1:1) birefringent 1,4- slurry(50° white solids;opaque B 1519- dioxane C.), 6 d masses of material; 73-02^(a) morphologyunknown slurry(50° small grainy solids; B 1557- C.), 2 d withbirefringence 12-01 subsample of — B 1557- 1557-12-01 15-01 subsample ofwhite solids B 1557- 1557-12-01 15-02 slurry(50° — B 1557- C.), 2 d17-01 isopropanol slurry(RT), white solids —^(b) 1519- 1 d 96-03tetrahydro- slurry(RT), — —^(b) 1519- furan 1 d 96-02 toluene slurry(50°white solids B 1519- C.), 6 d 73-01 ^(a)Seeds of sample #1480-58-01 (A +B) added ^(b)Samples not analyzed

Characterization of Crystal Forms: Form A

The initial material synthesized (termed Form A) was characterized usingX-ray powder diffraction (XRPD), differential scanning calorimetry(DSC), thermogravimetry (TG), hot stage microscopy, infrared (IR) andRaman spectroscopy, and moisture sorption. Representative XRPD of Form Ais shown in FIG. 15. The IR and Raman spectra for Form A are shown inFIGS. 16 and 17, respectively. The thermal data for Form A are shown inFIG. 18. The DSC thermogram shows an endotherm at 103° C. that isconsistent with melting (from hot stage microscopy). The sample wasobserved to recrystallize to needles on cooling from the melt. The TGdata shows no measurable weight loss up to 100° C., indicating that thematerial is not solvated. The moisture sorption data are showngraphically in FIG. 19. Form A material shows significant weight gain(>33%) during the course of the experiment (beginning between 65 to 75%RH), indicating that the material is hygroscopic. In addition,hygroscopicity of treprostinil diethanolamine was evaluated in humiditychambers at approximately 52% RH and 68% RH. The materials were observedto gain 4.9% and 28% weight after 23 days in the ˜52% RH and ˜68% RHchambers, respectively.

Based on the above characterization data, Form A is a crystalline,anhydrous material which is hygroscopic and melts at 103° C.

Form B

Treprostinil diethanolamine Form B was made from heated slurries (50°C.) of Form A in 1,4 dioxane and toluene, as shown in Table 16. Materialisolated from 1,4-dioxane was used to fully characterize Form B. Arepresentative XRPD pattern of Form B is shown in FIG. 20. Form A andForm B XRPD patterns are similar, however, significant differences areobserved in the range of approximately 12-17° 2θ (FIG. 20).

The thermal data for Form B are shown in FIG. 21. The DSC thermogram(Sample ID 1557-17-01) shows a single endotherm at 107° C. that isconsistent with a melting event (as determined by hotstage microscopy).The TG shows minimal weight loss up to 100° C.

The moisture sorption/desorption data for Form B are shown in FIG. 22.There is minimal weight loss at 5% RH and the material absorbsapproximately 49% water at 95% RH. Upon desorption from 95% down to 5%RH, the sample loses approximately 47%.

Form A and Form B can easily be detected in the DSC curve. Based on theabove characterization data, Form B appears to be a crystalline materialwhich melts at 107° C.

Thermodynamic Properties:

Inter-conversion experiments were carried out in order to determine thethermodynamically most stable form at various temperatures. Thesestudies were performed in two different solvents, using Forms A and Bmaterial, and the data are summarized in Table 17. Experiments inisopropanol exhibit full conversion to Form B at ambient, 15° C., and30° C. after 7 days, 11 days, and 1 day, respectively. Experiments intetrahydrofuran also exhibit conversion to Form B at ambient, 15° C.,and 30° C. conditions. Full conversion was obtained after 11 days at 15°C., and 1 day at 30° C. At ambient conditions, however, a minor amountof Form A remained after 7 days based on XRPD data obtained. Fullconversion would likely occur upon extended slurry time. Based on theseslurry inter-conversion experiments, Form B appears to be the mostthermodynamically stable form. Form A and Form B appear to be relatedmonotropically with Form B being more thermodynamically stable.

TABLE 17 Interconversion Studies of Treprostinil DiethanolamineExperiment/ Sample Starting Temper- No. Forms Solvent Materials atureTime 1557- A vs. B isopropanol solid mixture ambient 7 days 22-01 #1557-20-01^(a) 1557- A vs. B solid mixture 15° C. 11 days 47-02 #1557-35-01^(d) 1557- A vs. B solid mixture 30° C. 1 day 33-02 #1557-35-01^(d) 1557- A vs. B solid mixture 50° C. — 21-02^(e) #1557-20-01^(b) 1557- A vs. B tetrahydro- solid mixture ambient 7 days20-03 furan # 1557-20-01^(c) 1557- A vs. B solid mixture 15° C. 11 days47-01 # 1557-35-01^(d) 1557- A vs. B solid mixture 30° C. 1 day 33-01 #1557-35-01^(d) 1557- A vs. B solid mixture 50° C. — 21-01^(e) #1557-20-01^(c) ^(a)saturated solution Sample ID 1557-21-03 ^(b)saturatedsolution Sample ID 1519-96-03 ^(c)saturated solution Sample ID1519-96-02 ^(d)saturated solution prepared just prior to addition ofsolids ^(e)samples not analyzed as solubility (at 50° C.) oftreprostinil diethanolamine was very high and solutions becamediscolored.

All references disclosed herein are specifically incorporated byreference thereto.

While preferred embodiments have been illustrated and described, itshould be understood that changes and modifications can be made thereinin accordance with ordinary skill in the art without departing from theinvention in its broader aspects as defined herein.

1. A method of treating an ischemic disease comprising orallyadministering a pharmaceutical formulation comprising an effectiveamount of a compound of structure II to a subject in need thereof:

wherein, R¹ is independently selected from the group consisting of H,substituted and unsubstituted alkyl groups, arylalkyl groups and groups,wherein OR¹ form a substituted or unsubstituted glycolamide ester; R²and R³ may be the same or different and are independently selected fromthe group consisting of H, phosphate and groups wherein OR² and OR³ formesters of amino acids or proteins, with the proviso that all of R¹, R²and R³ are not H; an enantiomer of the compound; or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1, wherein when OR¹forms a substituted or unsubstituted glycolamide ester, R¹ is—CH₂CONR⁴R⁵, wherein R⁴ and R⁵ may be the same or different and areindependently selected from the group consisting of H, OH, substitutedand unsubstituted alkyl groups.
 3. The method of claim 2, wherein R⁴ andR⁵ are independently selected from —(CH₂)_(m)CH₃, and —CH₂(CH₂)_(n)OH,with the proviso that m is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3 or
 4. 4.The method of claim 1, wherein R¹ is a C₁-C₄ alkyl group.
 5. The methodof claim 4, wherein R¹ is selected from the group consisting of methyl,ethyl, propyl or butyl.
 6. The method of claim 1, wherein R¹ is asubstituted or unsubstituted benzyl group.
 7. The method of claim 1,wherein R¹ is —CH₃ or —CH₂C₆H₅.
 8. The method of claim 2, wherein R⁴ andR⁵ are the same or different and are independently selected from thegroup consisting of H, OH, —CH₃, and —CH₂CH₂OH.
 9. The method of claim1, wherein one or both of R² and R³ are H.
 10. The method of claim 1,wherein one or both of R² and R³ are not H and R² and R³ areindependently selected from phosphate and groups wherein OR² and OR³ areesters of amino acids, dipeptides, esters of tripeptides and esters oftetrapeptides.
 11. The method of claim 1, wherein only one of R² or R³is a phosphate group.
 12. The method of claim 10, wherein R² and R³ areindependently selected from groups wherein OR² and OR³ are esters ofamino acids.
 13. The method of claim 12, wherein one or both of R² andR³ are esters of glycine or alanine.
 14. The method of claim 10, whereinR¹ is H.
 15. The method of claim 10, wherein one of R¹ and R² is H. 16.The method of claim 14, wherein R² is H.
 17. The method of claim 1,wherein the oral bioavailability of the compound is greater than theoral bioavailability of treprostinil.
 18. The method of claim 17,wherein the oral bioavailability of the compound is at least 50% greaterthan the oral bioavailability of treprostinil as a free acid.
 19. Themethod of claim 18, wherein the oral bioavailability of the compound isat least 100% greater than the oral bioavailability of treprostinil. 20.The method of claim 1, further comprising administering pharmaceuticallyeffective amount of a p-glycoprotein inhibitor.
 21. The method of claim20, wherein the p-glycoprotein inhibitor is administered simultaneouslywith the compound of structure II.
 22. The method of claim 20, whereinthe p-glycoprotein inhibitor is administered prior to administration ofthe compound of structure II.
 23. The method of claim 20, wherein thep-glycoprotein inhibitor is administered orally or intravenously. 24.The method of claim 1, wherein the subject is a human.
 25. The method ofclaim 1, wherein the formulation is a sustained release formulation. 26.The method of claim 1, wherein the ischemic disease is Raynaud'sphenomenon.
 27. The method of claim 1, wherein the ischemic disease isScleroderma.
 28. The method of claim 1, wherein the ischemic disease isrenal insufficiency.
 29. A method of treating an ischemic diseasecomprising orally administering to a subject in need thereof apharmaceutical composition comprising an effective amount of a compoundof the following structure:


30. The method of claim 29, wherein the compound melts at about 107° C.31. The method of claim 30, wherein the compound has an x-ray powderdiffraction pattern having a pattern peak at about 17.2 degrees 2 theta.32. The method of claim 29, wherein the subject is a human.
 33. Themethod of claim 29, wherein the formulation is a sustained releaseformulation.
 34. The method of claim 29, wherein the formulation is asustained release capsule.
 35. The method of claim 29, wherein theformulation is a sustained release tablet.
 36. The method of claim 1,wherein the ischemic disease is Raynaud's phenomenon.
 37. The method ofclaim 1, wherein the ischemic disease is Scleroderma.
 38. The method ofclaim 1, wherein the ischemic disease is renal insufficiency.